A Comprehensive Review of Autophagy and Its Various Roles in Infectious, Non-Infectious, and Lifestyle Diseases: Current Knowledge and Prospects for Disease Prevention, Novel Drug Design, and Therapy

Abstract

Autophagy (self-eating) is a conserved cellular degradation process that plays important roles in maintaining homeostasis and preventing nutritional, metabolic, and infection-mediated stresses. Autophagy dysfunction can have various pathological consequences, including tumor progression, pathogen hyper-virulence, and neurodegeneration. This review describes the mechanisms of autophagy and its associations with other cell death mechanisms, including apoptosis, necrosis, necroptosis, and autosis. Autophagy has both positive and negative roles in infection, cancer, neural development, metabolism, cardiovascular health, immunity, and iron homeostasis. Genetic defects in autophagy can have pathological consequences, such as static childhood encephalopathy with neurodegeneration in adulthood, Crohn’s disease, hereditary spastic paraparesis, Danon disease, X-linked myopathy with excessive autophagy, and sporadic inclusion body myositis. Further studies on the process of autophagy in different microbial infections could help to design and develop novel therapeutic strategies against important pathogenic microbes. This review on the progress and prospects of autophagy research describes various activators and suppressors, which could be used to design novel intervention strategies against numerous diseases and develop therapeutic drugs to protect human and animal health.

Full text

cells
Review
A Comprehensive Review of Autophagy and Its
Various Roles in Infectious, Non-Infectious,
and Lifestyle Diseases: Current Knowledge and
Prospects for Disease Prevention, Novel Drug
Design, and Therapy
Rekha Khandia 1, Maryam Dadar 2, Ashok Munjal 1, * , Kuldeep Dhama 3, * ,
Kumaragurubaran Karthik 4, Ruchi Tiwari 5, Mohd. Iqbal Yatoo 6, Hafiz M.N. Iqbal 7,
Karam Pal Singh 3, Sunil K. Joshi 8, * and Wanpen Chaicumpa 9
1Department of Genetics, Barkatullah University, Bhopal 462 026, Madhya Pradesh, India
2Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension
Organization (AREEO), Karaj 31975/148, Iran
3Division of Pathology, ICAR-Indian Veterinary Research Institute, Izatnagar,
Bareilly 243 122, Uttar Pradesh, India
4Central University Laboratory, Tamil Nadu Veterinary and Animal Sciences University, Madhavaram Milk
Colony, Chennai, Tamil Nadu 600051, India
5Department of Veterinary Microbiology and Immunology, College of Veterinary Sciences, UP Pandit Deen
Dayal Upadhayay Pashu Chikitsa Vigyan Vishwavidyalay Evum Go-Anusandhan Sansthan (DUVASU),
Mathura, Uttar Pradesh 281 001, India
6
Sher-E-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar 190025,
Jammu and Kashmir, India
7Tecnologico de Monterrey, School of Engineering and Sciences, Campus Monterrey, Ave. Eugenio Garza
Sada 2501, Monterrey, N. L., CP 64849, Mexico
8Department of Pediatrics, Division of Hematology, Oncology and Bone Marrow Transplantation,
University of Miami School of Medicine, Miami, FL 33136, USA
9Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of
Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
*Correspondence: ak.munjal@bubhopal.ac.in (A.M.); kdhama@rediffmail.com (K.D.);
sunil.joshi@med.miami.edu (S.K.J.)
Received: 1 April 2019; Accepted: 4 June 2019; Published: 3 July 2019


Abstract:
Autophagy (self-eating) is a conserved cellular degradation process that plays important
roles in maintaining homeostasis and preventing nutritional, metabolic, and infection-mediated
stresses. Autophagy dysfunction can have various pathological consequences, including tumor
progression, pathogen hyper-virulence, and neurodegeneration. This review describes the mechanisms
of autophagy and its associations with other cell death mechanisms, including apoptosis,
necrosis, necroptosis, and autosis. Autophagy has both positive and negative roles in infection,
cancer, neural development, metabolism, cardiovascular health, immunity, and iron homeostasis.
Genetic defects in autophagy can have pathological consequences, such as static childhood
encephalopathy with neurodegeneration in adulthood, Crohn’s disease, hereditary spastic paraparesis,
Danon disease, X-linked myopathy with excessive autophagy, and sporadic inclusion body myositis.
Further studies on the process of autophagy in different microbial infections could help to design
and develop novel therapeutic strategies against important pathogenic microbes. This review on
the progress and prospects of autophagy research describes various activators and suppressors,
which could be used to design novel intervention strategies against numerous diseases and develop
therapeutic drugs to protect human and animal health.
Cells 2019,8, 674; doi:10.3390/cells8070674 www.mdpi.com/journal/cells

Cells 2019,8, 674 2 of 64
Keywords:
autophagy mechanism; autophagy-associated diseases; macroautophagy; chaperone-mediated
autophagy; apoptosis; necroptosis; necrosis; iron homeostasis; autophagy inhibition; AKT/mTOR signaling
pathway; autosis
1. Introduction
Autophagy is a conserved catabolic process that is involved in cellular homeostasis and is required
to maintain normal cellular physiology under stressful conditions [
1
]. It overcomes carcinogenic,
infectious, degenerative, and deleterious agents to maintain the homeostasis of bodily systems and
regulate healthy life processes; thus, its dysregulation is known to cause multiple human diseases [
2
–
5
].
Autophagy can be a selective or non-selective lysosomal degradative process and is activated by
stresses such as starvation or rapamycin via regulatory signaling complexes [
6
,
7
]. There are three types
of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA) [
8
].
Macroautophagy, referred to as “autophagy” from now on, is the major pathway which engulfs large
portions of cytoplasm and cellular contents (e.g., long-lived proteins, aggregated proteins, damaged
organelles, and intracellular pathogens) into a double-membraned vacuole called the autophagosome,
which fuses with lysosomes to form an autolysosome, degrades the autolysosomal contents, and recycles
macromolecules for reuse [9–11].
Microautophagy refers to the process by which lysosomes directly engulf and digest small volumes
of cytosolic substrate [
12
,
13
], whilst CMA is induced by physiological stresses such as prolonged
starvation [
14
] and involves the heat shock cognate protein (HSC70; 71-kDa, also known as HSPA8)
which contains a KFERQ-like pentapeptide sequence [
15
]. The CMA pathway delivers target proteins
across lysosomal membranes into the lysosomal lumen by interacting with lysosome-associated
membrane protein type 2A (LAMP-2A) [
9
]. Hence, CMA differs from microautophagy and
macroautophagy, as it does not require vesicular trafficking [
14
]. Regardless of the type, autophagy acts
as a cleaning mechanism by removing or degrading unnecessary materials from the body (e.g., proteins,
organelles, and microbes) and retaining or maintaining materials (biochemicals, metabolites,
and organelles) required for survival, function, and development [
1
,
6
,
16
,
17
]. The physiological
processes of autophagy are governed by numerous cellular regulators (e.g., transcription factors
and genes), which can affect homeostatic processing if disturbed by genetic or functional reasons,
or overexertion [4,6,17]. Hence, autophagy defects can affect the pathogenesis of many diseases [17].
The roles of autophagy have been explored in fields such as health, disease, infection, degeneration,
and genetic or lifestyle-acquired diseases [
18
–
21
]; however, cancer [
7
,
22
], microbial infections [
20
,
21
,
23
],
and degenerative diseases [
18
,
24
] have been the main focus of autophagy-related research. Currently,
the roles of autophagy are being explored in diverse fields of study.
Autophagy plays important roles in cancer metastasis; 4-acetyl-antroquinonol B has been shown
to modulate autophagy and prevent the growth of ovarian cancer cells [
25
]. Clinical studies have
revealed higher levels of autophagic flux in distant metastases than in primary tumors [
26
,
27
];
therefore, autophagy has stage-dependent dual roles in cancer which may facilitate the growth and
spread of tumors and affect treatment resistance [
19
]. Conversely, autophagy has been shown to act as
a tumor suppressor during the early and late phases of cancer development [
28
,
29
] by mediating the
destruction and removal of carcinogens and cancerous cells, thus enabling the growth and development
of healthy cells; however, under disturbed or uncontrolled conditions autophagy can promote cancer
growth and dissemination. Autophagy is also important in neuronal homeostasis, with its dysfunction
associated with numerous neurodegenerative disorders [
18
]. Pathogenic protein aggregates are a
common feature of neurodegenerative disorders, and dysfunctional autophagy is involved in this
disease state [
30
]. Furthermore, mutations in autophagy regulation genes have been shown to induce
neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer’s disease, and familial

Cells 2019,8, 674 3 of 64
Parkinson’s disease [
18
,
24
]. These mutations affect different stages of autophagy and thus have different
implications for pathogenesis and therapy [31].
In the modern world, factors such as globalization, liberal trade, climate change, population
explosions, public health lapses, immune pressures, and mutations, species jumping, and emerging
antibiotic resistance in pathogens have facilitated the spread of various infectious pathogens. In recent
years, it has been shown that autophagy has a role in many emerging and re-emerging infectious
viral and bacterial diseases that pose significant threats to humans. Autophagy initially encounters
these infectious pathogens to neutralize them; however, may infections can propagate themselves as
persistent intracellular infections and are generally associated with wide outbreaks, epidemics, and highly
devastating effects. Many viral life cycles are linked with autophagic pathways. The influenza A virus
induces autophagosome formation during the early stages of infection and inhibits autophagosomal
maturation during the later stages. Classical swine fever virus replication is negatively regulated by
mTORC1 via autophagy and IRES-dependent translation, whilst Dengue/Zika virus pathogenicity is
modulated by antibody-dependent enhancement (ADE) which can induce autophagy in human umbilical
vein endothelial cells. Multimodal necrotic cell death is driven by open reading frame-3a of severe acute
respiratory syndrome (SARS)-coronavirus (CoV), that triggers the lysosomal damage and dysfunction
and therefore transcription of autophagy-related genes is enhanced, whilst endoplasmic reticulum
(ER) stress in Dengue virus (DENV) infections results in autophagy activation, viral replication, and
pathogenesis. The disruption of mitochondrial membrane potentials by the non-structural protein of
Crimean-Congo hemorrhagic fever (CCHF) virus results in apoptosis, whereas paramyxovirus V proteins
inhibit constitutively active MDA5 proteins to induce autophagy. All of these viral events are related
to autophagy and can provide directions for future therapies for Chikungunya (CHIKV), DENV, and
Zika virus (ZIKV) infections. Autophagy has a pivotal role in viral diseases such as bird flu [
32
], swine
fever [
33
], Ebola virus disease [
20
], ZIKV infection [
34
,
35
], SARS [
36
], CHIKV infection [
37
], DENV
infection [
38
], viral encephalitis [
39
], CCHF [
40
], Hendra virus (HeV) infection [
41
], Nipah virus (NiV)
infection [
42
], and the West Nile virus (WNV) infection [
43
]. Apart from these, other viral diseases, such
as rabies, rotavirus enteritis, and smallpox, have already posed a serious threat to human life [
44
–
48
].
Autophagy has also been shown to have a central role in microbial infections [
49
], including those caused
by Listeria [
50
], Salmonella [
51
], Shigella [
52
], and Streptococcus [
53
]. Autophagy can kill or eradicate
infectious disease-causing pathogens via the autophagosome or autophagolysosome (autolysosome) to
prevent or treat infection [
20
,
21
]; however, autophagy can also disseminate pathogens during pathogenesis.
For example, gut epithelial autophagy can disseminate viruses and bacteria in enteric diseases. Therefore,
autophagy can play a dual role in infections [20,21,54].
In recent years, there has been an increase in the incidence of lifestyle and genetic diseases, such as
cancers and neurodegenerative disorders (Alzheimer’s, Parkinson’s, and Huntington’s diseases),
which affect the quality of life. Advances in science and technology have contributed to overcoming these
challenges. Novel, alternative, and complementary therapeutic options have been developed, including
phages, homing peptides, cytokines, siRNA, viral inhibitors, Toll-like receptors (TLRs), antibodies,
probiotics, herbs, phytomedicines, nanomedicines, and immunomodulatory techniques [
55
–
64
].
Autophagy is the first mechanism to clear endogenous debris and exogenous substances and maintains
normal physiological conditions in all eukaryotic cells [
65
]. Besides maintaining homeostasis [
66
],
autophagy also regulates the development [
67
], differentiation [
5
], and maturation [
68
] of cells,
such as endothelial cells [
69
], erythrocytes [
70
], and adipocytes [
71
,
72
]. These cells are involved in
normal physiological (e.g., erythrocytes in respiration), immunological (e.g., mononuclear cells in
immunity), metabolic (e.g., adipocytes in fat metabolism), growth (e.g., osteocytes in bone growth),
and development (e.g., spermatozoa or ova in reproduction) processes. Autophagy is also involved
in clearing abnormal protein accumulations and correcting mitochondrial disorganization
[73,74]
.
The processes of autophagy and apoptosis are interwoven and have been implicated in both
microbial infections [
54
,
75
] and cancers [
26
,
76
]. Autophagy might play both physiological and
pathological roles since it is involved in overcoming cell stresses [
19
,
77
,
78
]. Considering the numerous

Cells 2019,8, 674 4 of 64
roles and functions of autophagy in health and disease, we present a comprehensive overview
of autophagy, its mechanisms and types, and its associations with other cell death mechanisms.
The dual roles of autophagy in infectious diseases (bacterial and viral), tumor suppression/progression,
brain development/neurodegeneration, the immune system, and autoimmune diseases, and its other
roles have been discussed thoroughly alongside numerous applications of autophagy. We have also
summarized the role of autophagy in cardiovascular diseases, iron homeostasis, obesity, diabetes,
and diseases caused by defects in autophagy genes. The treatment of autophagy-associated diseases
has been described alongside strategies to inhibit or activate autophagy in the prevention and treatment
of diseases. This review details the important functions of autophagy in health and disease and its key
roles in disease prevention and treatment.
2. Autophagy: A Brief Overview
Autophagy (from the Greek words auto, meaning self, and phagy, meaning eating), is an essential,
ubiquitous, evolutionarily conserved, catabolic, and self-degradative process that mediates the
destruction of cytoplasmic macromolecules to preserve genomic integrity, achieve cell metabolism,
and ensure cell survival [
30
,
79
–
81
]. It is a natural regulatory mechanism which retains beneficial
substances and removes harmful substances from body, whilst playing a housekeeping role in the
elimination of misfolded or aggregated proteins, the eradication of damaged organelles, proteins [
82
–
84
],
and cancerous materials [
7
], and the elimination of foreign pathogens such as viruses via a degradative
lysosomal pathway [21,85–87].
Numerous physiobiological roles of autophagy have been identified, such as the disposal of
endogenous wastes and exogenous agents to maintain homeostasis; however, disturbing the natural
balance of this mechanism can result in pathological consequences [88].
Since it is the primary system for cleaning the body, autophagy can prevent or treat cancer
by killing cancerous cells and degrading endogenous or exogenous carcinogens; thus, favoring the
development of healthy cells. However, autophagy may have dual roles in cancer as it is involved in
stem cell-related resistance to anti-cancer therapy (radioresistance and chemoresistance), metastasis,
and tumor recurrence [
89
]. As obligate intracellular pathogens, viruses interact with multiple host
cell processes for their survival, including metabolism, cellular trafficking, and immunity-related
responses [
54
,
90
]. Furthermore, autophagy is a major degradative cellular process, with essential roles in
many innate and adaptive immune processes [
91
–
93
]. Autophagy also regulates the phosphorylation of
p38 and ERK1/2 MAPKs in BV2 microglial cells, required for nitric oxide production [
94
,
95
]. Thus, it can
affect the activation of neuronal cells by microglia and suppress neurotoxicity. Moreover, it can
downregulate pro-inflammatory mediators in BV2 microglial cells to rescue them from LPS- and
α-synuclein-induced neuronal cell death [94].
Autophagy can either be selective or non-selective [
96
]. In selective autophagy, cargo is recognized
by specific receptors to enable their specific identification, sequestration, and degradation by the
autophagosome, whereas in non-specific autophagy, all materials are degraded by the lysosome in
a non-specific manner [
96
,
97
]. Furthermore, autophagy is known to exist in two forms: constitutive
and reactive (induced) autophagy. Constitutive autophagy has not been well studied, whereas the
latter has been studied extensively and is known to stimulate neurite remodeling in developing
brains, thus may be essential during brain development [
98
,
99
]. Mice lacking the autophagy proteins
Atg59 and Atg710 display excessive neurodegeneration, indicating that autophagy has physiological
importance [
100
]. Numerous factors relating to nutrient deprivation (amino acids and hormones)
and build-up of degraded products (proteins) or exogenous agents (pathogens) have been evaluated
as induced autophagy stimuli [
101
,
102
]. Endogenous and exogenous stimuli induce autophagy for
degradation or as a repair mechanism.
Several stimuli have been shown to induce autophagy, including stress, amino acid starvation [
103
],
rapid declines in trophic factors or hormones (such as sex-based differences) [
104
], lipid starvation [
105
],
impaired intracellular cholesterol trafficking [
106
], protein products, and infectious pathogens [
32
,
38
,
85
].

Cells 2019,8, 674 5 of 64
These stimuli can affect the autophagic function and induce different morphological consequences
via diverse signaling pathways; for instance, suppressing phosphatidylinositol-3-kinase (PI3K)
inhibitors and Beclin 1 inhibits the starvation-induced mitochondrial autophagy, but not the neurotoxin
(1-methyl-4-phenylpyridinium)-mediated autophagy [
107
–
109
]. Although autophagy was discovered
over 50 years ago [
54
], its molecular mechanisms were only understood in the late 1990s following a
genetic screening in yeast, which revealed mutations in autophagy-related genes. At least 30 yeast
autophagy genes (Atgs) have been identified, many of which have mammalian cell homologs [85].
Many molecular mechanisms have been explored to reveal the basic processes underlying
autophagy. Multiple signaling pathways focus on two protein complexes to initiate autophagy,
the ULK1 (unc51-like autophagy activating kinase 1) protein kinase complex and the PI3KC3-C1
(class III phosphatidylinositol 3-kinase complex I) lipid kinase complex [
110
]. Novel autophagy
regulators with RNA-related activities have also been shown to be involved in this process [
111
].
Furthermore, upstream signaling pathways common to both autophagy and apoptosis are known to
be induced by ER stress via signaling molecules such as PERK/ATF4, IRE1
α
, ATF6, and Ca
2+
[
112
].
The details of these mechanisms will shed light on the different forms of autophagy and the numerous
intermediates involved.
Three types of autophagy [macroautophagy, microautophagy, and chaperone-mediated autophagy
(CMA)] are depicted in Figure 1.
Cells 2019, 8, x FOR PEER REVIEW 5 of 65
phosphatidylinositol-3-kinase (PI3K) inhibitors and Beclin 1 inhibits the starvation-induced
mitochondrial autophagy, but not the neurotoxin (1-methyl-4-phenylpyridinium)-mediated
autophagy [107–109]. Although autophagy was discovered over 50 years ago [54], its molecular
mechanisms were only understood in the late 1990s following a genetic screening in yeast, which
revealed mutations in autophagy-related genes. At least 30 yeast autophagy genes (Atgs) have been
identified, many of which have mammalian cell homologs [85].
Many molecular mechanisms have been explored to reveal the basic processes underlying
autophagy. Multiple signaling pathways focus on two protein complexes to initiate autophagy, the
ULK1 (unc51-like autophagy activating kinase 1) protein kinase complex and the PI3KC3-C1 (class
III phosphatidylinositol 3-kinase complex I) lipid kinase complex [110]. Novel autophagy regulators
with RNA-related activities have also been shown to be involved in this process [111]. Furthermore,
upstream signaling pathways common to both autophagy and apoptosis are known to be induced by
ER stress via signaling molecules such as PERK/ATF4, IRE1α, ATF6, and Ca2+ [112]. The details of
these mechanisms will shed light on the different forms of autophagy and the numerous
intermediates involved.
Three types of autophagy [macroautophagy, microautophagy, and chaperone-mediated
autophagy (CMA)] are depicted in Figure 1.
Figure 1. Different types of autophagy. Macroautophagy, microautophagy, and chaperone-mediated
autophagy.
2.1. Mechanisms of Autophagy
Autophagy refers to the process of delivering cytoplasmic or extracellular components to the
lysosomes of an animal cell or the vacuoles of plant or yeast cells [113]. The production and
maturation of autophagosomes are directly regulated by location, timing, and intensity [114]. The
phosphoinositide-binding protein, HS1BP3, is a negative regulator of autophagosome biogenesis that
regulates the lipid composition and phosphatidic acid (PA) levels of autophagosome precursor
membranes [114]. Increased levels of systemic autophagy have been reported in Caenorhabditis
Figure 1.
Different types of autophagy. Macroautophagy, microautophagy, and chaperone-mediated
autophagy.
2.1. Mechanisms of Autophagy
Autophagy refers to the process of delivering cytoplasmic or extracellular components to
the lysosomes of an animal cell or the vacuoles of plant or yeast cells [
113
]. The production
and maturation of autophagosomes are directly regulated by location, timing, and intensity [
114
].

Cells 2019,8, 674 6 of 64
The phosphoinositide-binding protein, HS1BP3, is a negative regulator of autophagosome biogenesis
that regulates the lipid composition and phosphatidic acid (PA) levels of autophagosome precursor
membranes [
114
]. Increased levels of systemic autophagy have been reported in Caenorhabditis
elegans, with hormetic heat stress and heat-shock responsive transcription factor (HSF-1) inducing
autophagy to improve the survival and proteostasis of the worm [
115
]. Furthermore, it has been
revealed that autophagy is fine-tuned by epigenetic regulation, through histone (coactivator-associated)
arginine methyltransferase, CARM1, a novel enzyme that follows histone H3R17 dimethylation
(histone H3 methylated at arginine 17) which is an important epigenetic marker of starvation-induced
autophagy [
116
]. In addition, the vitamin D receptor has been shown to modulate autophagy in
normal mammary glands and luminal breast cancer cells, suggesting a potential therapeutic link
between vitamin D levels and breast cancer risk [
117
]. There are numerous additional endogenous and
exogenous factors that modulate autophagy, such as transcription factors, variation in the amount
or concentration of various cytoplasmic biochemicals, damaged organelles, exogenous compounds,
and pathogens [
6
,
40
,
103
]; therefore, autophagy mechanisms vary. Autophagy can be divided into
macroautophagy, microautophagy, and CMA based on the mechanism by which intracellular materials
are delivered into the lysosome for degradation and the molecular structures that target substrates to
the lysosomes [
3
,
118
–
120
]. Although these pathways are mechanistically distinct, they all carry out
degradation via the lysosome [
54
,
118
]. Most forms of selective autophagy involve the degradation of
specific targets; for example, mitophagy (mitochondria), pexophagy (peroxisomes), aggrephagy (protein
aggregates), glycophagy (glycogens), lipophagy (lipids), ribophagy (ribosome), xenophagy (pathogens),
and ER-phagy [
21
,
121
]. Autophagy is a novel, evolutionarily conserved function of the eukaryotic
initiation factor 2 (eIF2
α
) kinase pathway, which consists of a family of evolutionarily conserved
serine/threonine kinases that regulate stress-induced translational arrest and are targeted by virulence
gene products [122].
2.1.1. Macroautophagy
Macroautophagy is initiated when a portion of cytoplasm containing a cellular organelle is sequestered
to form the autophagosome [
83
]. The autophagosome fuses with the lysosome or late endosomal
multivesicular bodies (MVBs) to degrade the materials within it. Atg8 (microtubule-associated protein
1A/1B-light chain 3, LC3, is an Atg8 homolog in humans) was the first autophagosomal protein to be
characterized [119]. Macroautophagy can be classified as cargo-specific or non-selective [83,119,123].
Mitophagy has been observed in yeasts when a shift occurs between non-fermentable and
fermentable carbon sources, such as glucose, following which the surplus mitochondrial population
undergoes mitophagy [
120
,
124
]. The first protein identified to cause mitophagy in yeast was Uth1p,
a member of the SUN family, which is present in the outer mitochondrial membrane and allows excessive
mitochondria to be removed during starvation [
125
]. The mitochondrial outer membrane protein,
Atg32, is a receptor for selective autophagy [
126
] that is not conserved in mammalian species; instead,
FUNDC1 and BNIP3, BNIP3L/NIX, and SQSTM1/p62 act as mitophagy receptors, and are dependent
upon hypoxia, erythrocyte maturation, and damage-induced mitophagy, respectively [
123
,
127
,
128
].
Pexophagy is also induced in Saccharomyces cerevisiae and Pichia pastoris via the Atg36 and PpAtg30
receptors, respectively, when the fungal medium is switched from an oleic acid or methanol to a glucose
or nitrogen starvation medium [
129
,
130
]. Starvation has also been shown to induce non-selective
macroautophagy [
9
], whereas mitochondrial phospholipids have been demonstrated to be required for
autophagy [
17
]. The machinery required for selective autophagy has been studied extensively using
yeast cells, revealing that the cytoplasm-to-vacuole targeting (CVT) pathway is used to specifically
transport vacuolar hydrolases into the vacuole of budding yeast cells [
131
]. A high degree of curvature
in the initiating membranes (phagophores or isolation membranes) is a prominent feature of CVT
vesicles during mammalian autophagy [132].

Cells 2019,8, 674 7 of 64
2.1.2. Microautophagy
After the lysosome has formed vesicles by invaginating and engulfing small sections of the
cytoplasm, lysosomal proteases degrade the contents of these vesicles [
119
]. Microautophagy occurs
during the biogenesis of multi-vesicular bodies (MVBs), which deliver soluble proteins to the late
endosomes, and relies on electrostatic interactions between endosomal sorting complexes required for
transport (ESCRT) I and III and the heat-shock cognate protein 70 (HSC70). Hence, microautophagy
involves both endocytic and autophagic components [133,134].
2.1.3. Chaperone-Mediated Autophagy (CMA)
Only proteins with a C-terminal pentapeptide KFERQ motif undergo CMA; the HSC70 cochaperone
identifies cytosolic proteins containing this sequence and delivers them to the lysosome [
135
,
136
].
Chaperones bound to the substrate are transported to the lysosomal surface, where they interact with
the monomeric LAMP-2A [
137
,
138
]. LAMP-2A must form a multiprotein complex to translocate the
substrate [
139
]; LAMP-2A complex assembly is a dynamic process that occurs when the substrate
binds to the receptor. The unfolded substrate protein (chaperon-mediated) is then translocated into the
lysosome by LAMP-2A for degradation, following which LAMP-2A disassembles and its monomers
are degraded in lipid microdomains. The levels of LAMP-2A tightly regulate the rate of CMA at
the lysosomal membrane [
15
,
140
]. In the mammalian anti-viral defense system, a cell-autonomous
autophagy mechanism has been identified wherein cellular p62 adaptor-mediated autophagic viral protein
clearance induces cell survival [
141
]. Some positive-strand RNA viruses, including picornaviruses and
influenza virus, promote autophagic membrane formation, and inhibit their final maturation (lysosomal
fusion) [
142
–
144
]. Consequently, studying the interactions between autophagy and adenoviruses could
improve adenoviral-based oncolytic virotherapies [145]. The process of CMA is depicted in Figure 2.
Cells 2019, 8, x FOR PEER REVIEW 7 of 65
2.1.2. Microautophagy
After the lysosome has formed vesicles by invaginating and engulfing small sections of the
cytoplasm, lysosomal proteases degrade the contents of these vesicles [119]. Microautophagy occurs
during the biogenesis of multi-vesicular bodies (MVBs), which deliver soluble proteins to the late
endosomes, and relies on electrostatic interactions between endosomal sorting complexes required
for transport (ESCRT) I and III and the heat-shock cognate protein 70 (HSC70). Hence,
microautophagy involves both endocytic and autophagic components [133,134].
2.1.3. Chaperone-Mediated Autophagy (CMA)
Only proteins with a C-terminal pentapeptide KFERQ motif undergo CMA; the HSC70
cochaperone identifies cytosolic proteins containing this sequence and delivers them to the lysosome
[135,136]. Chaperones bound to the substrate are transported to the lysosomal surface, where they
interact with the monomeric LAMP-2A [137,138]. LAMP-2A must form a multiprotein complex to
translocate the substrate [139]; LAMP-2A complex assembly is a dynamic process that occurs when
the substrate binds to the receptor. The unfolded substrate protein (chaperon-mediated) is then
translocated into the lysosome by LAMP-2A for degradation, following which LAMP-2A
disassembles and its monomers are degraded in lipid microdomains. The levels of LAMP-2A tightly
regulate the rate of CMA at the lysosomal membrane [15,140]. In the mammalian anti-viral defense
system, a cell-autonomous autophagy mechanism has been identified wherein cellular p62 adaptor-
mediated autophagic viral protein clearance induces cell survival [141]. Some positive-strand RNA
viruses, including picornaviruses and influenza virus, promote autophagic membrane formation,
and inhibit their final maturation (lysosomal fusion) [142–144]. Consequently, studying the
interactions between autophagy and adenoviruses could improve adenoviral-based oncolytic
virotherapies [145]. The process of CMA is depicted in Figure 2.
Figure 2. In chaperone-mediated autophagy (CMA), (1) KFERQ motif that is present in 30% of soluble
cytosolic proteins (2) is recognized by cytosolic chaperone protein HSPA8/HSC70, which is present in
a complex with other chaperone proteins. (3) Such recognized proteins bound to lysosomal receptor
Figure 2.
In chaperone-mediated autophagy (CMA), (
1
) KFERQ motif that is present in 30% of soluble
cytosolic proteins (
2
) is recognized by cytosolic chaperone protein HSPA8/HSC70, which is present in a
complex with other chaperone proteins. (
3
) Such recognized proteins bound to lysosomal receptor
protein LAMP-2A. (
4
) Binding of the substrate with the LAMP-2A leads to oligomerization of receptors.
(
5
) With the help of HSP90, the substrate is then unfolded and translocated through LAMP-2A-enriched
translocation complex. (
6
) After reaching inside the lysosomes, the proteins are degraded (
7
), and the
LAMP-2A receptors are disassembled.

Cells 2019,8, 674 8 of 64
2.2. Molecular Mechanisms of Autophagy
Autophagy is an evolutionarily conserved process induced via multiple signaling pathways by
numerous stimuli including nutrient starvation [
16
,
105
], hypoxia [
82
,
146
], oxidative stress [
109
,
147
],
pathogen infection [
39
,
148
], and ER stress [
149
]. In the presence of nutritional substances and
cytokines, mechanistic/mammalian target of rapamycin (mTOR) can prevent apoptosis and stimulate
cell growth [
150
], whereas stress and nutrient starvation inhibit mTOR to initiate autophagy via
at least four molecular complexes, including the unc-51-like kinase (ULK) complex, consisting of
ULK-1, Atg13, Atg101, and FAK-family interacting protein (FIP200); the PI3K complex, consisting
of Atg15, vacuolar protein sorting (VPS)15, VPS34, Beclin 1, and Beclin 1-regulated autophagy
protein 1 (AMBRA1) [
151
–
153
]; transmembrane protein complexes, including Atg9 and WIPI; and two
ubiquitin-like protein conjugation systems (Atg12 and LC3) [154,155].
Autophagy is initiated by the assembly of the ULK complex, which phosphorylates AMBRA1
and leads to activation of the PI3K complex [
155
,
156
]. Class III PI3K is known to participate in
various membrane trafficking events, whilst PI3K and Beclin 1 mediate membrane nucleation.
The Atg5-Atg12-Atg16 complex is recruited to the pre-autophagosomal structure (PAS) where
it associates with the outer membrane of the phagophore, essentially preventing the premature
fusion of vesicles and lysosomes [
157
]. The second ubiquitin-like system stimulates the binding of
phosphatidylethanolamine (PE) and Atg8/microtubule-associated protein 1 light chain 3 (LC3). LC3
has a high affinity for the lysosome when bound to the phagosome (LAPosome); thus, any engulfed
pathogens will be killed and degraded at a higher rate [
158
]. Atg4, Atg7, and Atg3 process
LC3 into LC3-II, a molecular marker for autophagosomes [
86
] that is present on both its
inner and outer surfaces and is essential for the expansion and completion of the autophagic
membrane. Following autophagosomal closure, the Atg5-Atg12-Atg16 complex dissociates from the
autophagosome. Atg9 is required for the formation of intraluminal vesicles and is localized within the
autolysosome for acidification [
159
]; Atg9 is also translocated to the site of autophagosome formation
where it provides a membrane to elongate the limiting membrane, known as the phagophore [
160
].
The autophagosome then fuses to the lysosome to form the autolysosome, which is regulated
by lysosomal membrane proteins and cytoskeletal proteins [
2
]. The LAMP-1/2 protein controls
autophagosomal maturation. Genetic mutations in LAMP-2 are known to cause Danon disease, a
glycogen storage disorder linked to hypertrophic cardiomyopathy, skeletal muscle weakness, and
intellectual disability [
161
]. Within the autolysosome, hydrolytic enzymes digest the internalized cargo
and the internal autophagosome membrane, then the digested products such as amino acids are released
into the cytosol to be recycled. Autophagosomes are also directly related to cell trafficking pathways.
Recently, Holland et al. identified that the phosphoinositide-binding protein HS1BP3 negatively
regulates autophagosome production [
162
]. HS1BP3 is thought to reduce phospholipase D1 (PLD1)
activity and its localization to ATG16L1 and transferrin receptor (TFRC)-positive vesicles. It is also
known to regulate the levels of PA and the lipid content of autophagosome precursor membranes [
114
].
Two large families of E3 ubiquitin ligases, TRIM and CULLIN, have been recognized as important
autophagy regulators which promote or inhibit the process, respectively [
81
]. The GTPase Ras-related
protein in brain 7 (Rab7) also plays a key role in autophagy regulation, particularly in modulating its
flux [
163
]. Knockdown of the small GTPase Rab13 has been shown to inhibit pterostilbene-induced
autophagy in vascular endothelial cells (VECs), whilst its upregulation stimulates autophagy in
VECs [
164
]. Under basal autophagy conditions in humans, proteomic analysis of the autophagy
interaction network (AIN) revealed a network of 751 interactions between 409 candidate proteins [
165
].
In order to identify the proteins modulating starvation-induced autophagy, genome-wide screening of
siRNA in a GFP-LC3-expressing human cell line was carried out [
166
], shortlisting nine proteins. One of
these, short coiled-coil protein (SCOC), forms an essential starvation-sensitive trimeric complex with UV
radiation resistance-associated gene (UVRAG) and WAC (WW domain-containing adapter protein with
coiled-coil), which is a negative regulator of the ubiquitin-proteasome system. Genome-wide studies in
C. elegans identified 139 genes that promote autophagy when inactivated [
167
]. Long ncRNAs (lncRNAs),

Cells 2019,8, 674 9 of 64
which are longer than 200 nucleotides and do not encode proteins, often possess regulatory functions; for
example, miR188-3 has been found to regulate Atg7 expression. RNA-linked strategies have revealed
several autophagy regulators such as RNA-binding proteins (RBPs), which are post-transcriptional
and co-translational regulators with RNA-related functions. Surprisingly, various key autophagy
proteins, including LC3B and LAMP-2C, have been found to bind RNA [
111
]. A considerable amount
of autophagy research is being carried out worldwide; however, these innovative findings have
raised numerous additional questions. To some extent, autophagy research has been protein-centric,
and innovative new approaches have been developed to strengthen this focus in recent years.
Among these, genome-wide screens and proteomics-based strategies have revealed substantial
interlinking between autophagy and RNAs; however, the precise mechanisms underlying this
association require further investigation. Future studies must develop and evaluate novel agents that
specifically target the autophagy pathway.
A pictorial representation of the process of autophagosome formation is presented in Figure 3.
Cells 2019, 8, x FOR PEER REVIEW 9 of 65
starvation-sensitive trimeric complex with UV radiation resistance-associated gene (UVRAG) and
WAC (WW domain-containing adapter protein with coiled-coil), which is a negative regulator of the
ubiquitin-proteasome system. Genome-wide studies in C. elegans identified 139 genes that promote
autophagy when inactivated [167]. Long ncRNAs (lncRNAs), which are longer than 200 nucleotides
and do not encode proteins, often possess regulatory functions; for example, miR188-3 has been
found to regulate Atg7 expression. RNA-linked strategies have revealed several autophagy
regulators such as RNA-binding proteins (RBPs), which are post-transcriptional and co-translational
regulators with RNA-related functions. Surprisingly, various key autophagy proteins, including
LC3B and LAMP-2C, have been found to bind RNA [111]. A considerable amount of autophagy
research is being carried out worldwide; however, these innovative findings have raised numerous
additional questions. To some extent, autophagy research has been protein-centric, and innovative
new approaches have been developed to strengthen this focus in recent years. Among these, genome-
wide screens and proteomics-based strategies have revealed substantial interlinking between
autophagy and RNAs; however, the precise mechanisms underlying this association require further
investigation. Future studies must develop and evaluate novel agents that specifically target the
autophagy pathway.
A pictorial representation of the process of autophagosome formation is presented in Figure 3.
Figure 3. Process of autophagosome formation. (1) Autophagy is inhibited by mTOR. (2) Various
kinds of stress (hypoxia, oxidative stress, pathogen infection, endoplasmic reticulum stress or nutrient
starvation conditions) inhibit mTOR, and the process of autophagy is initiated. (3) Assembly of ULK
complex occurs, and the complex includes ULK-1, autophagy-related protein 13 (Atg13), Atg101 and
FAK-Family Interacting Protein (FIP200). (4) The complex phosphorylates AMBRA1. (5) AMBRA1
activates PI3K complex encompassing Atg15, vacuolar protein sorting 15 (VPS15), VPS34, Beclin-1
and AMBRA1 which helps in nucleation. (6) Atg5-Atg12-Atg16 complex is recruited to phagophore
and prevent premature fusion of vesicle to the lysosome. (7) LC3 is conjugated with PE by the
ubiquitin-like system and (8) transformed into LC3-II with the help of Atg4, Atg7, and Atg3. (9) LC3-
II is present on both the inner and outer surfaces of the autophagosome. (10) Atg 9 further elongates
Figure 3.
Process of autophagosome formation. (
1
) Autophagy is inhibited by mTOR. (
2
) Various
kinds of stress (hypoxia, oxidative stress, pathogen infection, endoplasmic reticulum stress or nutrient
starvation conditions) inhibit mTOR, and the process of autophagy is initiated. (
3
) Assembly of ULK
complex occurs, and the complex includes ULK-1, autophagy-related protein 13 (Atg13), Atg101 and
FAK-
F
amily
I
nteracting
P
rotein (FIP200). (
4
) The complex phosphorylates AMBRA1. (
5
) AMBRA1
activates PI3K complex encompassing Atg15, vacuolar protein sorting 15 (VPS15), VPS34, Beclin-1 and
AMBRA1 which helps in nucleation. (
6
) Atg5-Atg12-Atg16 complex is recruited to phagophore and
prevent premature fusion of vesicle to the lysosome. (
7
) LC3 is conjugated with PE by the ubiquitin-like
system and (
8
) transformed into LC3-II with the help of Atg4, Atg7, and Atg3. (
9
) LC3-II is present on
both the inner and outer surfaces of the autophagosome. (
10
) Atg 9 further elongates the membrane
and forms intraluminal vesicles; also required for local acidification. (
11
) Atg5-Atg12-Atg16 complex is
dissociated from the complete autophagosome (12).

Cells 2019,8, 674 10 of 64
2.3. Autosis: A Novel Form of Autophagy
Liu and Levine [
168
] described a novel form of non-apoptotic autophagic gene-dependent cell
death, termed autosis, which is mediated by the Na
+
/K
+
-ATPase pump. Autosis involves enhanced
cell-substrate adhesion, focal ballooning of the perinuclear space, and dilation and fragmentation of
the endoplasmic reticulum. The Tat-Beclin 1 peptide complex may initiate autosis, with the fusion of
the evolutionarily conserved, 18-amino acid-long Beclin 1 domain with 11 amino acids from the HIV
Tat protein transduction domain aiding the cellular entry of the fusion peptide [
169
]. The Tat-Beclin 1
fusion peptide has been shown to inhibit the replication of HIV, CHIKV, Sindbis (SINV), and WNV,
as well as intracellular bacteria such as Listeria monocytogenes [
23
]. Tat-Beclin 1 treatment also reduced
mortality in neonatal mice infected with CHIKV and WNV, demonstrated using a TUNEL assay,
and cleared mutant Huntingtin protein aggregates [
170
]. Autosis can be partially rescued by knocking
down Atg13 or Atg14 or using 3-methyladenine. Under serum/amino acid starvation, approximately
1% of dying cells exhibit a morphology similar to that of cells treated with Tat-Beclin 1 and autosis
is selectively blocked when the Na
+
/K
+
-ATPase pump is inhibited [
171
]. During cerebral hypoxia
or ischemia, the neonatal brain releases cardiac glycosides (ouabain or endobain), which inhibit the
Na
+
/K
+
-ATPase pump and reduce autosis [
172
]. Autosis has also been observed in patients with severe
liver anorexia nervosa who display focal ballooning of the perinuclear space, convoluted nucleus,
dilated and fragmented ER, empty vacuoles, and several autolysosomes in their hepatocytes [
173
].
Ischemic injury can also lead to autosis in other organs, including the kidney and heart, which is
attenuated in Beclin 1+/−mice [168,174].
2.4. Association between Autophagy and Other Cell Death Mechanisms
Autophagy can promote or inhibit cell death depending on the cellular context; many other
death mechanisms are intricately involved in the processes, with several mechanistic links elucidated
between autophagy and other death mechanisms.
2.4.1. Links between Autophagy and Apoptosis
Autophagy and apoptosis regulation overlap when the BH3 domain of the Beclin 1
autophagy protein interacts with anti-apoptotic proteins of the Bcl-2 family, including Bcl-2, Bcl-xL,
and Mcl-1
[175–178]
. The BH3 domain has a critical role in the interaction between these proteins and
has been shown to interact with the receptor domain of the Bcl-2 family in nutrient-rich cells [
178
].
Beclin 1-mediated autophagy is inhibited by ER-localized Bcl-2 [
179
]; the transgenic expression of
Bcl-2 was shown to inhibit autophagy in mouse heart muscles. Beclin 1 mutants, which are unable
to bind to Bcl-2, induce higher levels of autophagy than their wild-type counterparts [
179
,
180
];
hence the physical Beclin 1-Bcl-xL/Bcl-2 interaction regulates Beclin 1-mediated autophagy [
179
].
ABT737, a compound which mimics the BH3 domain and thus inhibits this interaction, increases
the aggregation of LC3, an autophagy marker which is present on autophagosomes [
181
], in both
nutrient-rich and nutrient-deprived media. Furthermore, the knockdown of Beclin 1 and other
essential Atg proteins using siRNA heteroduplexes was shown to reduce ABT737-stimulated LC3
aggregation [
168
]. Atg12 is a dual-functioning protein that participates in both autophagy and
apoptosis [
108
]; non-conjugated Atg12 can bind and inhibit Mcl-1 and Bcl-2 via a BH3-like domain
to positively regulate mitochondrial apoptosis. Atg12 knockout inhibits the release of cytochrome c
from the mitochondria and apoptosis, whilst abnormal Atg12 expression represses the anti-apoptotic
activity of Mcl-1 [182].
Autophagy promotes apoptosis by degrading a negative regulator of the Fas ligand [
183
];
however, it can also protect cells against apoptosis induced by tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL) by altering the concentrations of Bcl family members [
184
].
Similarly, components were found to be degraded by autophagy during developmental apoptosis [
185
],
whilst it was recently shown that inhibiting autophagy increased apoptosis and accelerated mortality

Cells 2019,8, 674 11 of 64
in murine sepsis models with inadequate autophagy pathways in CD4
+
T cells, indicating that
autophagy has a functional role against apoptosis and immunosuppression in T cells in sepsis [
186
].
Furthermore, TRAIL combined with a novel chalcone derivative, Chal-24, was found to remarkably
increase lung cancer cell cytotoxicity via autophagy-mediated apoptosis [187].
2.4.2. Autophagy and Necroptosis
Necroptosis is often associated with inflammation [
63
]. The relationship between autophagy
and necroptosis is complex, elusive, and slightly controversial since reports have indicated that
necroptosis may promote [
188
], inhibit [
189
,
190
], or do not affect autophagy [
191
]. In several cell
lines, including L929 cells, lymphocytes, and cancer cells, autophagy is activated in the presence
of TNF
α
and under starvation to suppress necroptosis [
190
]. The apoptosis-inhibiting peptide,
carbobenzoxy-Val-Ala-Asp (zVAD), prevents autophagy and induces necroptosis in response to TNF
α
by regulating lysosomal cathepsins, highlighting the pro-survival function of autophagy against
necroptosis [
192
,
193
]. Similarly, inhibiting the mTOR signaling pathway can prevent apoptosis
and even enhance necroptosis, whereas starvation, which induces autophagy, protects cells from
zVAD-mediated necroptotic death [194].
Sirtuins (SIRT) are NAD
+
-dependent protein deacetylases which are actively involved in both
autophagy and necroptosis, as well as transcription, stress resistance, and aging. SIRT-1 deacetylates
various components of the autophagy pathway, including Atg5, Atg7, and Atg8 [
195
], thus promoting
autophagy. In cancer cells, dissociation of the FoxO1 transcription factor from SIRT-2 during oxidative
stress or starvation results in the acetylation and binding of FoxO1 to Atg7, which subsequently
induces autophagy [
196
]. The binding of SIRT-2 to receptor-interacting protein (RIP) 3 mediates
RIP1 deacetylation in response to TNF
α
; RIP1 and RIP3 then form a complex, which triggers
necroptosis [
197
]. The switch between necroptosis and apoptosis is achieved by recruiting necrosome
components to autophagy machinery. Atg5 knockdown reduced the association between RIPK1 and
MLKL, suggesting that Atg5 is important in TRAIL-induced necrosome activation. Furthermore, Atg5
knockout in the Atg5
-/-
DF-1 cell line inhibited autophagy but promoted apoptosis [
198
]. Autophagy
machinery also affects the mechanism of cell death by promoting efficient necrosomal activation
and MLKL phosphorylation, thus inducing necroptosis [
199
]. Several anti-cancer agents, including
sorafenib, cause deficient autophagosome formation and facilitate the interaction between p62 and
RIPK, resulting in cell death by necroptosis [
200
]; however, there is still much to be elucidated about
the interplay between these two processes.
2.4.3. Autophagy and Necrosis
Necrosis refers to the increase in cell volume caused by organelle swelling, which results in plasma
membrane rupture and the loss of intracellular contents. When ATP is depleted, the cell is unable
to undergo apoptosis and undergoes necrosis instead [
201
]. Poly ADP ribose polymerase (PARP1)
is an enzyme with roles in DNA repair, transcriptional regulation, and chromatin modification [
202
].
PARP1 over-activation decreases the ATP reservoir and induces necrotic cell death by bypassing
energy-dependent apoptotic cell death [
203
]. ATP depletion also activates AMP-activated kinases
(AMPK) [
204
], which induce autophagy by activating the ULK1 complex or inhibiting mTOR
signaling [
205
]. Thus, DNA damage-induced PARP1 activation leads to a decline in ATP levels, AMPK
activation, mTOR inhibition, and autophagy induction [
206
]. PARP1 plays a dual role in autophagy
and necrosis since autophagy is a pro-survival mechanism, whilst necrosis is a pro-death mechanism.
The fate of the cell depends on the balance between autophagy and necrosis, where autophagy
represents the final attempt of the cell to survive before necrosis.

Cells 2019,8, 674 12 of 64
3. Role of Autophagy
3.1. Role of Autophagy against Infectious Diseases
3.1.1. Anti-Bacterial Role of Autophagy
Autophagy plays a beneficial role against infectious diseases by simultaneously degrading
pathogens and activating the host immune system [
91
]. This enables infections to be countered directly,
by killing infectious agents, and indirectly, by inducing host immunity against pathogens. Autophagy
provides an excellent intracellular defense system against bacterial pathogens, including Salmonella
enterica serovar Typhimurium [
51
,
207
], Listeria monocytogenes [
50
,
208
], and Shigella flexneri [
209
].
Anti-bacterial autophagy is termed xenophagy [
21
,
53
,
210
]. Numerous cellular, membrane-associated,
or cytoplasmic moieties modulate xenophagy; and those cells unable to carry out xenophagy, exhibit
higher rates of infection. Bcl-xL regulates xenophagy, and Bcl-xL knockout cells are more susceptible
to Streptococcus pyogenes infection [
53
]. The infection of non-phagocytic cells by Shigella flexneri is
dependent upon type-III secretion system (T3SS) effector proteins [
52
] which reorganize the host cell
cytoskeleton, ruffle the cell membrane, and cause bacterial uptake. Following internalization, bacterial
peptidoglycans are detected by nucleotide-binding oligomerization domain (NOD)-like receptors
(NLRs) which trigger a pro-inflammatory immune response [
211
] (Figure 4). The bacteria-sensing
NOD proteins interact with Atg16L1 to initiate anti-bacterial autophagosome biogenesis in response
to bacterial invasion [
212
]. Intracellular bacterial sensing either by NLRs or sequestosome-1-like
receptors (SLRs) recruits autophagy proteins, including unc-51-like kinase (ULK) 1/2 and lipid kinase
complexed with Beclin 1 and Atg16L1, to initiate phagophore membrane nucleation and engulf
invading bacteria [213].
Mutant C. elegans with defective autophagy genes exhibit increased susceptibility to bacterial
infection [
214
]. In addition, it has been reported that HLH-30/TFEB-mediated autophagy and autophagy
pathways can regulate the tolerance of C. elegans to Bacillus thuringiensis infection by protecting against
its pore-forming toxins [
215
], suggesting a novel association between intrinsic epithelial defenses and
HLH-30-mediated autophagy against
in vivo
bacterial attacks. Liang et al. [
176
] reported that Beclin 1
overexpression inhibits Sindbis virus replication, indicating that autophagy protects against infectious
pathogens. Autophagy may activate innate immunity against mycobacteria via pattern recognition
receptors (PRRs) or non-receptor-mediated processes [49].
Infection with group A Streptococcus species (GAS; Streptococcus pyogenes) induces anti-apoptotic
Bcl-xL expression which inhibits autophagy directly by suppressing autophagosome-lysosome fusion,
and indirectly by interacting with Beclin 1-UVRAG to suppress GAS internalization [
53
]. In addition,
Mycobacterium tuberculosis is known to induce miR144 expression in human macrophages and monocytes
and adversely affect their antimicrobial activities and innate host immune responses against the bacterial
infection by targeting DRAM2 (DNA damage-regulated autophagy modulator 2), which is a critical
element of the autophagy response [
216
]. The ubiquitin ligase, SMURF1, has also been shown to
control M. tuberculosis replication in human macrophages by associating with bacteria in the lungs of
patients with pulmonary tuberculosis. The murine macrophage cell line, RAW264.7, has been used
to study Bacillus amyloliquefaciens SC06-induced autophagy and its anti-bacterial response against
Escherichia coli;B. amyloliquefaciens stimulated autophagy by increasing the expression of Beclin 1 and
the Atg5-Atg12-Atg16 complex, but not activating the AKT/mTOR signaling pathway [217].
Several autophagy-inducing drugs have been used to treat microbial infections; for example,
AR-12 [2-amino-N-[4-[5-(2 phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] phenyl]-acetamide]
inhibits phosphoinositide-dependent kinase-1 and eliminates Salmonella typhimurium in murine
macrophages and Francisella tularensis in human leukemic THP-1 macrophages [
218
,
219
]. Furthermore,
1
α
,25-dihydroxycholecalciferol, a form of vitamin D, can enhance autophagy and inhibit human
immunodeficiency virus (HIV) replication in macrophages [220].

Cells 2019,8, 674 13 of 64
Cells 2019, 8, x FOR PEER REVIEW 12 of 65
3. Role of Autophagy
3.1. Role of Autophagy against Infectious Diseases
3.1.1. Anti-Bacterial Role of Autophagy
Autophagy plays a beneficial role against infectious diseases by simultaneously degrading
pathogens and activating the host immune system [91]. This enables infections to be countered
directly, by killing infectious agents, and indirectly, by inducing host immunity against pathogens.
Autophagy provides an excellent intracellular defense system against bacterial pathogens, including
Salmonella enterica serovar Typhimurium [51,207], Listeria monocytogenes [50,208], and Shigella flexneri
[209]. Anti-bacterial autophagy is termed xenophagy [21,53,210]. Numerous cellular, membrane-
associated, or cytoplasmic moieties modulate xenophagy; and those cells unable to carry out
xenophagy, exhibit higher rates of infection. Bcl-xL regulates xenophagy, and Bcl-xL knockout cells
are more susceptible to Streptococcus pyogenes infection [53]. The infection of non-phagocytic cells by
Shigella flexneri is dependent upon type-III secretion system (T3SS) effector proteins [52] which
reorganize the host cell cytoskeleton, ruffle the cell membrane, and cause bacterial uptake. Following
internalization, bacterial peptidoglycans are detected by nucleotide-binding oligomerization domain
(NOD)-like receptors (NLRs) which trigger a pro-inflammatory immune response [211] (Figure 4).
The bacteria-sensing NOD proteins interact with Atg16L1 to initiate anti-bacterial autophagosome
biogenesis in response to bacterial invasion [212]. Intracellular bacterial sensing either by NLRs or
sequestosome-1-like receptors (SLRs) recruits autophagy proteins, including unc-51-like kinase
(ULK) 1/2 and lipid kinase complexed with Beclin 1 and Atg16L1, to initiate phagophore membrane
nucleation and engulf invading bacteria [213].
Figure 4. Anti-bacterial role of autophagy. (1) Bcl-xL regulates the autophagy, and in Bcl-xL knockout
cells, Streptococcus pyogenes infection is promoted. (2) Shigella flexneri invasion in non-phagocytic cells
is dependent upon the type-III secretion system (T3SS) effector proteins. Following internalization
nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) detect bacterial
Figure 4.
Anti-bacterial role of autophagy. (
1
) Bcl-xL regulates the autophagy, and in Bcl-xL
knockout cells, Streptococcus pyogenes infection is promoted. (
2
)Shigella flexneri invasion in
non-phagocytic cells is dependent upon the type-III secretion system (T3SS) effector proteins. Following
internalization nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) detect bacterial
peptidoglycans and trigger pro-inflammatory immune response. Bacterial sensing inside the cell either
by NLRs or sequestosome-1-like receptors (SLRs) recruits autophagy proteins including unc-51-like
kinase (ULK) 1/2 with lipid kinase complexed with Beclin 1 and Atg16L1 to initiate membrane nucleation
of the phagophore to engulf invading bacteria. (
3
) Group A Streptococcus species inhibits autophagy
directly by suppressing the fusion of autophagosomes. (
4
)Bacillus amyloliquefaciens was found to
stimulate autophagy by elevating the expression of Beclin 1 and Atg5-Atg12-Atg16 complex.
Many bacteria have evolved mechanisms to overcome autophagy and allow them to replicate
within infected cells or even within autophagosomes. These bacteria may express receptors to prevent
or enhance phagosome formation, capture nutrient containing phagosomes, subvert autophagy
machinery, prevent fusion, or resist autophagy. Certain bacteria hijack autophagosomes and use the
by-products of autophagic degradation for microbial replication [
221
]. Anaplasma (formerly Ehrlichia)
phagocytophilum, the causative agent of human anaplasmosis, uses the effector Anaplasma translocated
substrate 1 (Ats-1) to enhance autophagosome formation and acquire nutrients from inside the
autophagosome [
222
]. After entering the cell, A. phagocytophilum replicates inside a double-lipid bilayer
membrane associated with LC3 (Atg8), Beclin 1, and Atg6 but lacking lysosomal markers. Inhibiting
autophagy with 3-methyladenine did not prevent bacterial internalization but arrested its growth [
223
],
indicating that the autophagic machinery had been subverted to facilitate bacterial proliferation.
Another bacterium, Yersinia pseudotuberculosis, replicates intracellularly inside specific compartments
called Yersinia-containing vacuoles (YCVs), which contain autophagy markers; however, YCVs are
not acidified and sustain bacterial replication [
224
]. During Y. pestis infection, LC3-I is conjugated
with PE to recruit LC3-II, a marker of autophagy progression, to the phagosomal membrane [
225
].
A similar mechanism is used by Coxiella burnetii, the causative organism of Q fever. Coxiella-replicative

Cells 2019,8, 674 14 of 64
vacuoles contain LC3, Beclin 1, and Rab24; overexpression of these proteins increases the number
of Coxiella-replicative vacuoles [
226
]. Brucella abortus replicates within Brucella-containing vacuoles
(BCVs) which traffic from the endocytic compartment to the endoplasmic reticulum, where the
bacteria proliferate. Bacterial proliferation requires the autophagy-initiation proteins ULK1, Beclin 1,
and Atg14L; however, Atg5, Atg16L1, Atg4B, Atg7, and LC3B are not required [227].
Pathogens such as Brucella spp. and Porphyromonas gingivalis have evolved to survive inside
autophagosomes by preventing its fusion with the lysosome, thus escaping host innate immunity
mechanisms [
228
,
229
]. Salmonella typhimurium studies have revealed that autophagy targets invading
intracellular bacterial pathogens for degradation [
230
]; S. typhimurium regulates the SIRT1/LKB1/AMPK
complex of the mTOR pathway by targeting SIRT1, LKB1, and AMPK to lysosomes for rapid
degradation, restricting autophagy and disrupting AMPK-mediated mTOR regulation [231].
Autophagy is differentially regulated in tuberculoid and lepromatous leprosy [
232
]; in tuberculoid
skin lesion cells, autophagy controls Mycobacterium leprae, whereas in lepromatous cells, the blocking of
Bcl-2-mediated autophagy promotes bacterial persistence. IFN-
γ
may counteract the M.leprae–mediated
inhibition of autophagy in lepromatous macrophages as autophagy levels were restored in lepromatous
patients who developed the reversal reaction, an inflammatory state associated with augmented IFN-
γ
and rapamycin treatment, indicating that autophagy is an important innate mechanism associated
with M.leprae control in skin macrophages [232].
3.1.2. Anti-Viral Role of Autophagy
Autophagy has a beneficial role in cellular defense against invasion by viruses; therefore, it has been
used for antiviral immunity [
141
,
233
,
234
]. Autophagy helps to clear viral pathogens during infection
via various molecular mechanisms, regulates immune responses, and prevents harmful overactivation
and inflammation [
235
]. For example, autophagy increases the presentation of endogenous viral
antigens in the peptide grooves of major histocompatibility complex (MHC) class I molecules on the cell
surface during herpes simplex virus type 1 (HSV-1) infection. Studies of viral peptides have suggested a
complex interaction between vacuoles and MHC class I presentation pathways in autophagosomes [
236
].
In contrast, MHC class II molecules continuously accept input from autophagosomes, which facilitates
antigen presentation by MHC class II molecules [
237
,
238
]. Autophagy is a major component of
Drosophila immunity against vesicular stomatitis virus (VSV) [
239
] as it can deliver viral antigens to
TLRs for presentation. During anti-viral signaling, pattern recognition receptors (PRRs) at the plasma
membrane (i.e., Toll-7) that are engaged by VSV stimulate an autophagy-dependent innate immune
response mediated by PI3K-Akt-signaling [
239
,
240
]. Furthermore, Toll/TLR signaling has been shown
to regulate the Rift Valley fever virus (RVFV) replication in both flies and mammals [
241
], whilst SIRT1,
an NAD(+)-dependent deacetylase, modulates the activation of dendritic cells and autophagy during
induced immune responses against respiratory syncytial virus (RSV) [
242
], thereby directing an
effective anti-viral immune response. Furthermore, autophagy is stimulated by the salicylamide
derivatives against cytopathic bovine viral diarrhea virus (cp-BVDV), a Flaviviridae pestivirus [
243
].
Foot-and-mouth disease virus (FMDV) infection suppresses autophagy and NF-
κ
B anti-viral activities by
degrading Atg5-Atg12 using the viral protein, 3C
pro
, suggesting that Atg5-Atg12 positively modulates
anti-viral NF-
κ
B and IRF3 pathways during FMDV infection to limit FMDV proliferation [
244
].
However, autophagy is often hijacked by viral pathogens and can be modulated to their own benefit.
3.1.3. Proviral Role of Autophagy
Subverting the autophagic pathway can have adverse consequences by giving pathogens access to
nutrients for growth and reproduction [
245
]. Autophagy plays an important role in viral replication and
pathogenesis [
246
], with coronaviruses [
247
], coxsackievirus B3 [
248
], poliovirus [
249
], hepatitis C virus
(HCV) [
142
,
250
–
252
], and DENV [
143
] all known to stimulate and require autophagy for accelerated
replication. For instance, autophagy has been demonstrated to be actively involved in the replication of
influenza A virus (IAV), which induces autophagosome formation during the early phase of infection

Cells 2019,8, 674 15 of 64
and later inhibits autophagosomal maturation by preventing autophagosomal-lysosomal fusion and
promoting autophagosomes to accumulate in virus-infected cells [
253
]. Autophagy-deficient cells
are more susceptible to apoptosis upon influenza infection [
253
,
254
], while using pharmacological
reagents or RNA interference to alter cellular autophagy can impair viral protein accumulation [
255
].
Human single-chain antibody variable fragments (ScFvs) which bind to the influenza A virus ion
channel protein (M2) and inhibit viral replication [
256
] were found to restore autophagosome maturation
suppressed by the infecting virus (personal communication). It has also been reported that HCV
can trigger autophagy via immunity-related GTPase M, which promotes HCV replication [
257
].
Paramyxoviruses such as Newcastle disease virus (NDV) have been shown to trigger autophagy in
U251 glioma cells to enhance viral replication [
258
]. In addition, modulating NDV-induced autophagy
using rapamycin, chloroquine, or small interfering RNAs which target genes critical for autophagosome
formation (Atg5 and Beclin 1) affects virus production, suggesting that NDV may utilize autophagy to
promote its replication [
259
]. Human immunodeficiency virus (HIV) uses multiple methods to regulate
autophagy and enhance its replication [
260
,
261
]. HIV induces the early stages of autophagy but inhibits
the later stages which would suppress the production of new virions. The HIV-1 accessory protein,
Nef, inhibits autophagosomal maturation by interacting with Beclin 1 [
262
], whilst the HIV protein Vpr
can trigger autophagy in transfected THP-1 macrophages, indicating that autophagy may be involved
in maintaining HIV reservoirs in macrophages [
263
]. HSV-1 [
264
], Kaposi’s sarcoma-associated
herpesvirus (KSHV) [
265
], and mouse herpesvirus 68 (MHV-68) encode proteins that bind Beclin 1 to
prevent autophagy initiation [266].
During poliovirus (PV) infection, vesicle acidification, which can mature autophagosomes, has been
shown to induce the maturation of virions into infectious particles [
267
]. One of the most important
characteristics of high-risk human papillomavirus (hrHPV) etiopathogenesis is that inhibiting host
autophagy could cause cervical cancer via hrHPV [
268
]. In epithelial cells, flavivirus NS4A-induced
autophagy protects infected cells and induces viral replication [
269
]. Autophagy also plays a critical role
in the replication of coronaviruses and the generation of their replicative structures [
270
]. Coronavirus
nonstructural proteins (nsp6) induce the formation of omegasomes and autophagosomes from the ER
via an omegasome intermediate [
271
]. In addition, autophagy has been shown to induce the replication
of infectious spleen and kidney necrosis virus (ISKNV) in the Chinese Perch Brain (CPB) cell line,
suggesting complex interactions between ISKNV and host cells during viral pathogenesis and for
anti-viral treatment strategies [246].
Treating FMDV-infected cells with rapamycin, an autophagy inducer, was shown to increase viral
replication, whilst inhibiting the autophagosomal pathway using 3-methyladenine or small-interfering
RNAs decreased viral replication [
272
]. Furthermore, disrupting autophagy using the knockdown
approach in hepatitis C virus (HCV)-infected hepatocytes stimulated the interferon signaling pathway
and induced apoptosis, indicating that HCV-induced autophagy can impair the innate immune
response [
251
]. Suppressing HCV-induced autophagy could be a promising approach for inhibiting
exosome-mediated viral transmission [
273
], besides autophagy has been shown to reduce HCV
clearance following IFN-
α
/Ribavirin (RBV)-based anti-viral therapy [
274
]. A DENV study revealed
that autophagy inhibitors are better candidate targets than conventional anti-viral therapies using
interferons (IFNs) [
275
]; upregulating cellular autophagy was reported to inhibit RLR-mediated type-I
IFN-independent signaling and cause the antibody-dependent enhancement (ADE) of DENV [
274
].
Suppressing autophagic vacuoles has been demonstrated to stimulate the maturation of infectious
bursal disease virus [
276
]. Adenoviral infection may be favored by autophagy via an increase in ATP,
essential to increase anabolism of the infected cells and amino acid pools for the synthesis of viral
proteins. In the later stages of adenoviral infection, Atg12-Atg5 complex is significantly upregulated as
an evidence of enhanced autophagy [
277
]; therefore, autophagy may improve the virulence of some
viruses. Autophagy genes are involved in the regulation and execution of autophagy [
278
]. Beclin 1
was the first mammalian gene identified to stimulate autophagy [
279
]. Some viruses, such as
α
-,
β
- and
γ
-herpesviruses, encode the neurovirulence protein, ICP34.5, which associates with Atg6/Beclin 1 and

Cells 2019,8, 674 16 of 64
inhibits autophagy by preventing the formation of the PI3 kinase complex [
264
]. The autophagy genes
Fip200,Beclin 1,Atg14,Atg16l1,Atg7,Atg3, and Atg5 have been found to promote the reactivation of
latent murine gamma-herpesvirus 68 by inhibiting virus-induced systemic inflammation molecules,
such as IFN-
γ
[
280
]. In contrast, autophagy inhibition has been reported as a new molecular mechanism
by which HSV-1 escapes innate immunity, resulting in fatal disease [
281
]. The autophagic cell death of
alveolar epithelial cells has been observed to play a major role in the high mortality rate caused by H5N1
influenza virus infection; hence autophagy-blocking agents could have preventative and therapeutic
effects against this virus [
282
]. Activating the PI3K /Akt/mTOR pathway and inhibiting autophagy
have been shown to promote the cellular entry of HPV type 16 [
283
], contrarily autophagy has been
shown to be essential for the replication of coronavirus and mouse hepatitis virus (MHV) [
284
]. HSV-1
mutants, those are unable to inhibit autophagy grows to low virus titer and are less pathogenic [
264
].
Autophagy evokes antiviral adaptive immunity via the endogenous presentation of viral antigens
through the MHC class II pathway [278].
The proviral and anti-viral actions of autophagy are illustrated in Figure 5.
Cells 2019, 8, x FOR PEER REVIEW 16 of 65
gamma-herpesvirus 68 by inhibiting virus-induced systemic inflammation molecules, such as IFN-γ
[280]. In contrast, autophagy inhibition has been reported as a new molecular mechanism by which
HSV-1 escapes innate immunity, resulting in fatal disease [281]. The autophagic cell death of alveolar
epithelial cells has been observed to play a major role in the high mortality rate caused by H5N1
influenza virus infection; hence autophagy-blocking agents could have preventative and therapeutic
effects against this virus [282]. Activating the PI3K /Akt/mTOR pathway and inhibiting autophagy
have been shown to promote the cellular entry of HPV type 16 [283], contrarily autophagy has been
shown to be essential for the replication of coronavirus and mouse hepatitis virus (MHV) [284]. HSV-
1 mutants, those are unable to inhibit autophagy grows to low virus titer and are less pathogenic
[264]. Autophagy evokes antiviral adaptive immunity via the endogenous presentation of viral
antigens through the MHC class II pathway [278].
The proviral and anti-viral actions of autophagy are illustrated in Figure 5.
Figure 5. Proviral and anti-viral actions of autophagy.
3.2. Autophagy in Tumor Suppression
Initially, autophagy was thought to be involved in tumor suppression by stimulating gene
expression, inhibiting proinflammatory mediators, inhibiting inflammation or inflammatory
products, and stimulating signaling pathways. The essential Atg6/Beclin 1 gene was found to be lost
monoallelically in 40–75 % of human prostate, breast, and ovarian cancers [285], whereas excessive
autophagy stimulation by Beclin 1 overexpression has been reported to inhibit tumor progression
[286,287]. Autophagy causes necrosis and chronic inflammation by inhibiting the release of pro-
inflammatory HMGB1, which is involved in tumorigenesis [288]. In cell-based assays, inhibiting
autophagy was shown to enhance cancer cell growth [289]. p62 (a signaling adaptor/scaffold protein)
is involved in the formation of intracellular ubiquitin-related protein aggregates because of
autophagy deficiency. Atg7-deficient mice exhibit enhanced accumulation of p62 and ubiquitinated
protein aggregates in hepatocytes and neuron [290]. Autophagy has also been implicated in benign
hepatomas [291], and the inactivation of Beclin 1 and Atg5 was shown to increase the incidence of
Figure 5. Proviral and anti-viral actions of autophagy.
3.2. Autophagy in Tumor Suppression
Initially, autophagy was thought to be involved in tumor suppression by stimulating gene
expression, inhibiting proinflammatory mediators, inhibiting inflammation or inflammatory products,
and stimulating signaling pathways. The essential Atg6/Beclin 1 gene was found to be lost monoallelically
in 40–75 % of human prostate, breast, and ovarian cancers [
285
], whereas excessive autophagy
stimulation by Beclin 1 overexpression has been reported to inhibit tumor progression [
286
,
287
].
Autophagy causes necrosis and chronic inflammation by inhibiting the release of pro-inflammatory
HMGB1, which is involved in tumorigenesis [
288
]. In cell-based assays, inhibiting autophagy was
shown to enhance cancer cell growth [
289
]. p62 (a signaling adaptor/scaffold protein) is involved in

Cells 2019,8, 674 17 of 64
the formation of intracellular ubiquitin-related protein aggregates because of autophagy deficiency.
Atg7-deficient mice exhibit enhanced accumulation of p62 and ubiquitinated protein aggregates
in hepatocytes and neuron [
290
]. Autophagy has also been implicated in benign hepatomas [
291
],
and the inactivation of Beclin 1 and Atg5 was shown to increase the incidence of cancer in mice [
292
].
Atg5- and Atg7-deficient mice exhibited liver tumors, indicating that defective autophagy can affect
the suppression of tumorigenesis [
292
]. Heterozygous Beclin 1 (beclin 1+/- mutant) was shown to
have a high incidence of spontaneous tumors [
289
,
293
], whilst Beclin 1 inhibited tumor growth in
cell lines such as the breast cancer cell line, MCF-7, in which Beclin 1 expression was lower than in
normal epithelial breast cells [
294
]. The UVRAG protein was found to suppress the tumorigenicity and
proliferation of human colonic cancer cells [295] (Figure 6).
Cells 2019, 8, x FOR PEER REVIEW 17 of 65
cancer in mice [292]. Atg5- and Atg7-deficient mice exhibited liver tumors, indicating that defective
autophagy can affect the suppression of tumorigenesis [292]. Heterozygous Beclin 1 (beclin 1+/-
mutant) was shown to have a high incidence of spontaneous tumors [289,293], whilst Beclin 1
inhibited tumor growth in cell lines such as the breast cancer cell line, MCF-7, in which Beclin 1
expression was lower than in normal epithelial breast cells [294]. The UVRAG protein was found to
suppress the tumorigenicity and proliferation of human colonic cancer cells [295] (Figure 6).
Figure 6. Autophagy in tumor suppression. (1) The Atg5- or Atg7-deficient mice showed liver tumors,
indicating that defective autophagy can affect the suppression of tumorigenesis. (2) Beclin 1 inhibits
the growth of tumor in cell lines such as the breast cancer cell line, MCF-7, in which the expression of
Beclin 1 was lower than in normal epithelial breast cells. (3) UVRAG protein could suppress
tumorigenicity and proliferation of colon cancer cells in humans. (4) mTOR is implicated in cancer
and its substrates include the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) and the
ribosomal S6 kinases (S6Ks) 1 and 2, which promote cell cycle progression. The mTOR, which is
inhibited by rapamycin, induces autophagy. (5) A novel anti-cancer molecule, HA15, which targets
HSPA5/BIP was shown to induce endoplasmic reticulum stress and increase the unfolded protein
response, resulting in cancer cell death through autophagy and apoptosis.
Autophagy suppresses tumor formation by preventing inflammation, the accumulation of
proteins and organelles damaged by necrosis, and cellular transformation caused by gene instability
[296–299]. The conserved protein kinase, mTOR, has been implicated in cancer since its substrates
(eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) and ribosomal S6 kinases (S6Ks) 1
and 2) promote cell cycle progression [299]. mTOR, which is inhibited by rapamycin [300], and
molecules such as phosphatase and tensin homolog (PTEN) and tuberous sclerosis (TSC) (products
of tumor suppressor genes) can induce autophagy [301,302]. Pogostone, a medicinal herb widely used
to treat gastrointestinal diseases, was shown to possess anti-colorectal tumor activities by stimulating
autophagy and apoptosis via the PI3K/Akt/mTOR axis [303]. In addition, the novel anti-cancer
molecule HA15, which targets HSPA5/BIP, was shown to induce ER stress and increase the unfolded
Figure 6.
Autophagy in tumor suppression. (
1
) The Atg5- or Atg7-deficient mice showed liver tumors,
indicating that defective autophagy can affect the suppression of tumorigenesis. (
2
) Beclin 1 inhibits the
growth of tumor in cell lines such as the breast cancer cell line, MCF-7, in which the expression of Beclin
1 was lower than in normal epithelial breast cells. (
3
) UVRAG protein could suppress tumorigenicity
and proliferation of colon cancer cells in humans. (
4
) mTOR is implicated in cancer and its substrates
include the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) and the ribosomal S6
kinases (S6Ks) 1 and 2, which promote cell cycle progression. The mTOR, which is inhibited by
rapamycin, induces autophagy. (
5
) A novel anti-cancer molecule, HA15, which targets HSPA5/BIP was
shown to induce endoplasmic reticulum stress and increase the unfolded protein response, resulting in
cancer cell death through autophagy and apoptosis.
Autophagy suppresses tumor formation by preventing inflammation, the accumulation of proteins
and organelles damaged by necrosis, and cellular transformation caused by gene instability [
296
–
299
].
The conserved protein kinase, mTOR, has been implicated in cancer since its substrates (eukaryotic
initiation factor 4E (eIF4E)-binding proteins (4E-BPs) and ribosomal S6 kinases (S6Ks) 1 and 2)

Cells 2019,8, 674 18 of 64
promote cell cycle progression [
299
]. mTOR, which is inhibited by rapamycin [
300
], and molecules
such as phosphatase and tensin homolog (PTEN) and tuberous sclerosis (TSC) (products of tumor
suppressor genes) can induce autophagy [
301
,
302
]. Pogostone, a medicinal herb widely used to
treat gastrointestinal diseases, was shown to possess anti-colorectal tumor activities by stimulating
autophagy and apoptosis via the PI3K/Akt/mTOR axis [
303
]. In addition, the novel anti-cancer molecule
HA15, which targets HSPA5/BIP, was shown to induce ER stress and increase the unfolded protein
response, resulting in cancer cell death via autophagy and apoptosis [
304
]. Trichosanthin (TCS),
a 27 kDa protein from the bioactive component of the root tuber of Trichosanthes kirilowii (Chinese
cucumber plant; Gua Lou in Mandarin), also exhibited anti-cancer properties against different human
ovarian cancer cells via a pathway common to both autophagy and apoptosis [305].
3.3. Autophagy in Tumor Progression
Various factors and mechanisms are involved in autophagy-mediated tumor progression.
Tumor-induced nutrient shortage, cell debris (degraded proteins), inflammation, and oxidative
cascades are all molecular mechanisms affecting tumor progression. During nutrient starvation,
autophagy induction promotes the survival of normal cells and may also promote tumor cell survival;
however, hypoxia, metabolic stress, energy shortage, oxidative stress-damaged mitochondria, and
organelles can be caused by cancer-causing genes or cancer treatments [
306
]. The undifferentiated
colon cancer cell line, HT-29, and other transformed cells have shown an increased tendency to degrade
autophagic proteins [
287
,
307
,
308
]. The elevated expression of the autophagy signature protein, BNIP3,
a pro-apoptotic Bcl-2 member, has been demonstrated in colorectal and gastric epithelial carcinomas,
suggesting that BNIP3 expression may be important for the development of these cancers [
309
].
The activation of autophagy and peroxisome proliferator-activated receptor gamma (PPAR
γ
) was
shown to protect colon cancer cells against apoptosis induced by the interaction between butyrate
and docosahexaenoic acid (DHA) in a cell type-dependent manner [
310
]. Additionally, an Atg8/LC3
family member implicated in autophagy and tumor suppression was associated with alterations
to cell death and cytokine secretion in mice lacking gamma-aminobutyric acid receptor-associated
protein (GABARAP) [
311
]. It has been well documented that inhibiting autophagy in cancer cells
increases their death, with this strategy proving most useful in tumors that behave like RAS-activated
tumors [
312
]. Inhibiting autophagy is expected to cause ubiquitinated proteins to accumulate and p62
levels to increase; in hepatic tumors, autophagy suppresses spontaneous tumorigenesis via cell-intrinsic
pathways whilst p62 accumulation promotes tumor formation [291].
The elimination of damaged organelles via autophagy may allow cancer cells to survive despite
the stress caused by chemotherapeutic agents [
313
]. It has also been reported that upregulating
autophagy after chemotherapy causes cancer cells to enter a dormant state, which may then propagate
at a later stage [
314
,
315
]. The state of cell cycle arrest, termed senescence, has been postulated to
underlie autophagy-induced tumor cell dormancy [
316
]. Ras-induced senescence is mediated by
autophagy, with autophagy inhibition delaying senescence [
317
]. Moreover, it has been reported that
PSMD10/gankyrin stimulates autophagy in hepatocellular carcinoma (HCC) in response to starvation
or stress. A physical association occurs between PSMD10 and Atg7, and is translocated to the nucleus
to bind to ATG7 promote and upregulates Atg7 expression [318].
3.4. Autophagy in Brain Development
There is growing evidence that autophagy performs both physiological and pathological functions
in the nervous system, and that autophagic stimulation plays critical roles in neuronal survival and
activity. Autophagy is the only method by which neurons degrade and excrete expired organelles,
and is responsible for clearing abnormal intracytoplasmic contents from normal cells which would
otherwise cause protein accumulation and damage neuronal activity, inducing severe functional
impairment. Autophagy also clears protein aggregates from old neurons; thus, inhibiting autophagy
can lead to neuronal degeneration and intraneuronal protein accumulation. Mutations in Atg5 confined

Cells 2019,8, 674 19 of 64
to neural tissues can cause impaired growth, progressive motor and behavioral deficits, prominent
neurodegeneration, and axonal swelling in regions of the brain with increased levels of ubiquitinylated
proteins, indicating that autophagy has a neuroprotective role [
319
]. In mammals, the absence of
Atg59 and Atg710 can cause severe neurodegeneration, further supporting the neuroprotective role of
autophagy [
100
,
320
]. Furthermore, neuronal death can be attributed to the loss of basal autophagy or an
imbalance in autophagic flux. In some neurodegenerative diseases, such as Alzheimer’s, Parkinson’s,
and Huntington’s, as well as in the brain or spinal cord trauma, the damaged neurons exhibit
abnormally high numbers of autophagosomes. Therefore, understanding the interaction between
pathophysiological mechanisms and autophagy could be a promising approach for therapies against
neurological disorders [
321
]. Prenatal alcohol exposure has been shown to increase the number of
autophagic vacuoles in the cortical micro-vessels of human fetal and mouse neonatal brains, impairing
autophagy [
322
]. Furthermore, autophagy can modulate Notch degradation, stem cell development,
and neurogenesis [323].
3.5. Autophagy in Neurodegeneration
Several reviews have evaluated the relationship between autophagy and neurodegenerative
diseases [
324
,
325
]. Autophagy is vital for neuronal homeostasis [
326
], and its deregulation is highly
associated with numerous neurodegenerative effects, such as the accumulation of damaged and
toxic molecules with pathological consequences in neurodegenerative disorders such as Alzheimer’s,
Parkinson’s, and Huntington’s diseases [
327
,
328
]. Lysosomal system inactivation is responsible for
the accumulation of autophagosomes observed in Alzheimer’s disease [
329
], whilst the disease is
thought to be due to either excessive or impaired autophagosomal degradation, or the activation
of autophagy genes in response to temporary injury/stress in neuronal tissues. Alzheimer’s [
330
],
Parkinson’s
[331,332]
, and Huntington’s diseases [
333
] are key examples where autophagosomal
accumulation and anomalies in the endosomal-lysosomal pathway have been observed in post-mortem
human brain tissues via electron microscopy. Autophagy deficiency resulted in neuronal loss
in the cerebral and cerebellar cortices in a mouse model [
334
]. Dysfunction or abnormalities in
autophagy, including mutations in autophagy-regulating genes, are accompanied by neurodegenerative
diseases across the age spectrum with exceptional frequency. Atg7-deficient mice exhibited
ubiquitin accumulation in their CNS, causing nervous symptoms, neurodegeneration, and ultimately
death [
31
,
334
], whilst Atg5-deficient mice developed cytoplasmic inclusions and exhibited motor
dysfunctions [319], and AMBRA 1-deficient mouse embryos displayed neuronal tube defects [335].
Autophagy is involved in the cytoplasmic clearance of
α
-synuclein (
α
-syn), which is observed
in Parkinson’s disease [
336
]. In a mouse model, Beclin 1 overexpression was found to reduce the
clearance of
α
-syn, leading to pathological neuron abnormalities [
337
]. Pharmacological and genetic
pathways are involved in the degradation of
α
-syn by polo-like kinase 2 via autophagy, suggesting
that these two proteins are concomitantly co-degraded [338].
The PINK1 and Parkin genes regulate mitophagy [
339
], indicating that mutations in these genes
can cause defects in mitophagy which have been correlated with Parkinson’s disease [
340
,
341
]. Beclin 1
expression is lower in the brains of patients with Alzheimer’s disease and not only affects autophagy
but also increases the deposition of
β
-amyloid proteins causing neurodegeneration [
330
]. Huntington’s
disease is caused by the extension of the polyglutamine (polyQ) proteins aggregate intracellularly,
which causes neuronal death. Atg-knockout C. elegans exhibit increased polyQ toxicity [
342
], whilst in
Drosophila the autophagy-enhancing small molecule 2-(4-phenylphenyl)-5,6-dihydroimidazo[2,1-B][1,3]
thiazole, also known as autophagy enhancer-99 (AUTEN-99), has been shown to prevent the symptoms
of neurodegenerative diseases [
343
]. The dual role observed for autophagy may be due to our poor
understanding of this ubiquitous cellular recycling system. The differences between physiological
and pathological autophagy may help design therapeutic strategies specifically targeting pathological
autophagy without hindering its physiological roles [
344
]. For example, the apoptosis-stimulating
protein p53-2 (ASPP2/53BP2L) was reported to have different effects on autophagy in neurons stimulated

Cells 2019,8, 674 20 of 64
with different levels of gp120, a soluble envelope glycoprotein of HIV-1 that interacts with chemokine
receptors such as CXCR4 and CCR5. Thus, regulating autophagy in the CNS could be a potential
therapeutic approach against HIV-associated neurocognitive disorders [77].
3.6. Autophagy in the Immune System and Autoimmune Diseases
3.6.1. Autophagy in the Immune System
The roles of cellular autophagy in immunological processes and autoimmune diseases have been
reviewed extensively [
82
,
345
,
346
]. Autophagy plays important roles in both innate and adaptive
immunity [
347
], modulates cellular and humoral immune responses [
348
–
350
], and has roles in the
non-metabolic and metabolic functions of immune cells [
349
]. Furthermore, autophagy is involved
in innate immune cell differentiation, degranulation, phagocytosis and extracellular trap formation
involving neutrophils, eosinophils, mast cells, and natural killer cells, and plays an essential role in
the renewal, differentiation, and homeostasis of immune cells [
351
]. Autophagy also regulates the
functional responses of immune cells, such as phagocytosis, antigen presentation, cytokine production,
control of inflammasome activation, tolerance, and their consequences on overall host defense via
monocytes, macrophages, dendritic cells, and antigen presentation [
350
]. Additionally, autophagy
plays important roles in B cell development, activation, and differentiation, which enables B cells
to adapt to various events, and determines their fate, survival, and function [
352
]. Since B cells
produce antibodies, autophagy can determine humoral immune responses. In one study, the B cells of
Atg5-deficient mice had defective antibody responses, indicating that autophagy has a role in antibody
production [353].
Pro-Inflammatory Signaling Regulated by Autophagy
Several studies have documented interplay between autophagy and the NF-
κ
B signaling pathway.
Members of the NF-
κ
B family of transcription factors regulate the transcription of genes involved
in cell proliferation, survival, differentiation, and development, whilst activation of the inhibitor of
NF-
κ
B (I
κ
B
α
) kinase complex is essential for autophagy induction. T-cell receptor-mediated NF-
κ
B
activation in B-cell lymphoma/leukemia is linked with the autophagy adaptor p62/SQSTM1 [
354
],
which modulates the NLRP3-inflammasome activation and IL-1βproduction in macrophages [355].
Interplay between Cytokine Secretion and Autophagy
IL-1
α
secretion is enhanced in Atg5-deficient macrophages, whilst inhibiting autophagy results in
IL-1
β
overexpression [
356
]. The anti-inflammatory cytokine, IL-10, inhibits autophagy by activating
mTOR complex 1 [
357
] and inhibits starvation- and IFN-
γ
-induced autophagy via Bcl-2 and Beclin 1 in
various autoimmune and inflammatory disorders [358].
3.6.2. Autophagy and Autoimmunity
Autophagy has predisposing, pathogenic, and therapeutic roles in autoimmunity. Defects in
autophagy pathways and/or autophagy-related genes have been implicated in numerous autoimmune
and autoinflammatory diseases, including multiple sclerosis, systemic lupus erythematosus (SLE),
rheumatoid arthritis, psoriasis, psoriatic arthritis, inflammatory bowel disease, diabetes mellitus,
Crohn’s disease, and vitiligo [
82
,
358
–
360
]. Abnormalities in the maintenance of homeostasis via
autophagy result in the accumulation of dysfunctional or defective cellular organelles, abnormal
proteins, infectious agents, and metabolite accumulation. This predisposes cells to the generation of
autoantibodies and proinflammatory mediators and exposes vital and susceptible cellular structures to
deleterious agents that can cause disease [
358
–
361
]. A recent study revealed a correlation between the
expression pattern of autophagy-related genes and the type of lupus nephritis (LN); thus, autophagy
could indicate of the type of LN when formulating a treatment regimen [362].

Cells 2019,8, 674 21 of 64
Several Atgs are known to be involved in autoimmune disorders, including Atg5, PR domain zinc
finger protein 1 (PRDM1; also known as BLIMP-1), and DNA-damage regulated autophagy modulator
1 (DRAM1) in SLE patients and Atg16L1 and immunity-related GTPase M (IRGM) in Crohn’s disease
and ulcerative colitis. Autophagy defects have been observed in T cells, B cells, and macrophages [
363
].
MHC class II antigen presentation by macrophages occurs via CMA; lysosomal proteins have a
central role in antigen processing, which is essential for a correct immune system function. Studies in
MRL/lpr mice which develop a full panel of lupus autoantibodies revealed that increased lysosomal
pH might be an important lysosomal malfunction involved in autoimmunity, and that perturbed
lysosomal turnover may lead to hyperactive antigen presentation by antigen presenting cells (APC) in
autoimmune disorders [363].
In innate immunity, reduced Atg5 and mTOR expression result in defective autophagy, affecting
the clearance of dead cells, increasing levels of nucleic acid remnants and self-antigens, increasing type
1 IFN by DCs, and inducing B cell hyper-differentiation and autoantibody production [
82
,
346
,
358
].
It has also been reported that autophagy-related gene knockdown can have therapeutic effects on
autoimmune diseases [
357
]. Modulating autophagy can manage immunity-related and inflammatory
diseases [
82
,
345
,
347
,
364
] by regulating cytokine and antibody production against immunogenic insults
to prevent autoimmune diseases. Therefore, regulating autophagy has clinical potential in cancer
immunotherapy [
365
], whilst autophagy and adenoviral combinations are proving beneficial in
adenoviral-based oncolytic virotherapy [145].
3.7. Autophagy in Cardiovascular Diseases
Under normal conditions, the myocardium exhibits low levels of autophagy, whilst stressful
conditions can increase the level of autophagy to increase cell survival [
366
,
367
]. Patients with
congestive heart failure, coronary artery disease, hypertension, and aortic valvular disease display
increased autophagosomal accumulation in their myocardial biopsies [
368
]. Autophagy levels vary in
normal and affected or stressed hearts, with constitutive autophagy maintaining normal structure and
function, and upregulated autophagy occurring during cardiac disease or stress [
369
]. In Atg5-deficient
mice, contractile dysfunction and hypertrophy have been observed during cardiomyopathy [
370
],
whilst cell culture studies have revealed that autophagic gene deficiency can cause the accumulation
of unwanted proteins and contribute to myocardial disease [
371
]. Similarly, LAMP-2-deficient mice
displayed increased autophagic vacuole accumulation and could not degrade proteins, thereby
promoting cardiomyopathy [
371
,
372
]. It has been postulated that during early life, between birth and
suckling, autophagy provides the energy required for cardiac cells [
373
], whilst mitophagy protects
cardiac muscles under ischemic stress [374].
Increased autophagy can cause heart failure [
375
], with autophagy-induced degeneration resulting
in the death of cardiomyocytes. This knowledge has helped our understanding of the pathogenic role
of autophagy in cardiac failure models and helped devise therapeutic targets [
376
,
377
]. Autophagy can
cause myocardial cell damage via PARP1, which promotes autophagy in cardiomyocytes by modulating
FoxO3a transcription [
378
]. Increased autophagy causes pathological remodeling of the heart, whilst
decreased autophagy reduces remodeling [
88
]. Thus, it has both protective and destructive roles in the
cardiovascular system.
3.8. Autophagy in Iron Homeostasis
Iron homeostasis involves a form of macroautophagy known as ferritinophagy, wherein ferritin,
an iron storage protein, is degraded in the lysosome [
379
]. Iron levels are tightly regulated in cells;
nutrient deficiency induces autophagy, during which cellular proteins and organelles are engulfed
by the autophagosome, which then fuses with the lysosome. The degradation of these contents
provides essential resources that either promote cell survival or lead to cell death. Iron (Fe), copper
(Cu), zinc (Zn), and aluminum (Al) react with molecular oxygen to produce reactive oxygen species
(ROS) and reactive nitrogen species (RNS). Besides acting as a cofactor for metalloprotein enzymes

Cells 2019,8, 674 22 of 64
involved in redox reactions, iron also plays a major role in mitochondrial ATP metabolism and other
cellular processes. The Fenton and Haber-Weiss redox reaction is highly involved in ROS production
and Alzheimer’s progression [
380
]. To maintain iron homeostasis, storage and recycling are critical.
When engulfed by macrophages, the iron within erythrocytes is either stored as a ferritin complex or
exported from the cell by the ferroportin iron-exporter [381].
HSP70 and ferritin bind iron in the cytosol and autophagocytosis of these proteins can sequester
redox-active iron in the lysosomes [
382
]. Cells rich in these proteins exhibit increased resistance to
oxidative stress; therefore, autophagy plays a major role in maintaining cellular redox status [
383
,
384
].
The autophagy inhibitor NCOA4, which is a substrate of PI3K, has been shown to physically bind to
the ferritin protein complex and direct it to autolysosomes for degradation [
385
]. NCOA4 knockdown
prevents the localization of ferritin in the lysosomes and increases the levels of iron-responsive
element–binding protein 2 (IRP2), which is a free intracellular Fe antagonist that prevents cell death
via exogenous ROS [
386
]. NCOA4 also acts as an autophagy receptor for ferritin and delivers it to the
lysosome to maintain iron homeostasis. Experimentally simulating low-iron conditions by chelating
iron revealed that ferritin is degraded to release the stored iron [387].
3.9. Autophagy in Obesity and Diabetes
Autophagy is involved in obesity [
388
] and diabetes mellitus [
389
]. Improper lipid and glycogen
processing can affect the liver activity and thus, insulin synthesis, resulting in diabetes. Studies have
shown that hepatocytes from mouse models of obesity display reduced autophagy [
390
], with decreased
Atg7 expression causing ER stress and affecting insulin signaling [
390
]. Mutant Atg7 mice also
exhibit reduced
β
cell mass, reduced insulin circulation, and glucose intolerance, indicating that
autophagy defects can reduce insulin levels and cause hyperglycemia [
391
,
392
]. A genetic mosaic
screen for mutations that increase lysosomal and/or autophagic activity in D. melanogaster larva
revealed that autophagy-lysosome pathways underlie novel cytoprotective features in Drosophila [
393
].
Obesity impairs autophagy in the liver via S-nitrosylation, a process induced by nitric oxide (NO).
S-nitrosylation of the lysosomal enzymes cathepsin B (CTSB) and hexosaminidase subunit
β
(HexB)
impairs normal lysosomal functioning and is carried out by denitrosylation enzymes, particularly
S-nitrosoglutathione reductase (GSNOR) and thioredoxin [
394
]. Obesity inhibits the denitrosylation
ability of the liver, impairing hepatic autophagy and insulin resistance [
395
]. In obese animals, hepatic
insulin signaling is impaired by NO-induced hepatic autophagy repression, which ultimately causes
the progression of type 2 diabetes [396].
The potential roles of autophagy in ameliorating diseases while maintaining homeostasis have
been enumerated in Table 1. Details of applied/granted patents for treating autophagy-related ailments
and dysfunctions are presented in Table 2.

Cells 2019,8, 674 23 of 64
Table 1. Potential role of autophagy in ameliorating/deteriorating diseases and homeostasis.
S. No. Activity Associated
with Autophagy Effect of Autophagy Modus Operandi of Related Activity and Example/Proof of Concept Reference(s)
1Viral infection
Anti-viral activity
Endogenous viral antigen presentation on MHC class-1 in Herpes simplex virus type 1 (HSV-1)
infection English et al., 2009 [236]
Delivery of viral antigens to Toll-like receptors (TLRs)- in Vesicular stomatitis virus (VSV) infection;
Pattern recognition receptor Toll-7 mediated PI3K-Akt-signaling
Shelly et al., 2009 [239],
Nakamoto et al., 2012 [240]
Sirtuin 1, a NAD(+)-dependent deacetylase mediated dendritic cell and autophagy induction -
Respiratory syncytial virus (RSV) Owczarczyk et al., 2015 [242]
Autophagy by salicylamide derivates- anti-viral activity against - Cytopathic bovine viral diarrhea
virus (cp-BVDV) flavivirus Needs et al., 2016 [243]
Inhibition of Sindbis virus replication by overexpression of Beclin 1 Liang et al., 1998 [176]
Enhanced autophagy by 1α,25-dihydroxycholecalciferol reduces HIV replication Campbell and Spector, 2012
[220]
During foot and mouth disease virus infection, Atg5-Atg12 enhances NF-κB and IRF3 pathways Fan et al., 2017 [244]
Targeting glycoproteins E1 and E2 and non-structural proteins of Chikungunya virus (CHKV) Subudhi et al., 2018 [397]
Pro-viral activity
Rapamycin, chloroquine and small interfering RNAs target Atg5 and Beclin 1- virus production is
hampered in New Castle disease virus (NCDV) Sun et al., 2014 [259]
Induction of early stages of autophagy and inhibition of later destructive stages – to conquer
suppression of new virion production- HIV Kyei et al., 2009 [261]
Nef-mediated inhibition of maturation of autophagosome- HIV
NS4A-induced autophagy in epithelial cells induces virus replication – Flavivirus McLean et al., 2011 [269]
Limitation of autophagosomal by 3-methyladenine or small-interfering RNAs- diminished
replication of virus- FMDV O’Donnell et al., 2011 [272]
Virus-induced autophagy-mediated impairment of innate immune response- Hepatitis C virus
(HCV) Shrivastava et al., 2011 [252]
Diminished viral clearance by IFN-α/RBV-based antiviral therapy-HCV Dash et al., 2016 [274]
Inhibition of RLR-mediated type-I IFN-independent signaling resulting in antibody-dependent
enhancement (ADE) of Dengue virus (DENV) Huang et al., 2016 [275]
Adenoviral infection may be privileged by autophagy via an increase in ATP; Atg12-Atg5 complex
is significantly upregulated. Jiang et al., 2008 [277]
Activation of the phosphatidylinositol 3 kinase/Akt/mTOR pathway and inhibition of autophagy-
induce cellular entry of –Human Papilloma virus (HPV) type 16. Surviladze et al., 2013 [283]
Replication of Infectious Spleen and Kidney Necrosis virus (ISKNV) is increased when autophagy
is induced Li et al., 2017 [246]
Human nuclear ribonucleoprotein K (hnRNP-K) and ubiquilin 4 (UBQLN4) help in viral replication.
NDP52 human autophagy receptor interacts with CHIKV nsP2 and acts as proviral factor Wong and Chu, 2018 [37]

Cells 2019,8, 674 24 of 64
Table 1. Cont.
S. No. Activity Associated
with Autophagy Effect of Autophagy Modus Operandi of Related Activity and Example/Proof of Concept Reference(s)
Classical swine fever virus replication is negatively regulated through mTORC1 Luo et al., 2018 [33]
Autophagosomal targeting of ribosomal proteins by influenza A virus (IAV) Becker et al., 2018 [32]
Autophagy of endothelial cells of umbilical vein by Zika virus (ZIKV) helps in replication Peng et al., 2018 [35]
Necrosis of cells through severe acute respiratory syndrome-coronavirus (SARS-CoV) open reading
frame-3a for multiplication Yue et al., 2018 [36]
ER stress by DENV infection helps in autophagy and replication, both in vitro and in vivo Lee et al., 2018 [38]
Non-structural protein of virus affects mitochondrial membrane in Crimean-Congo Hemorrhagic
fever causing apoptosis Barnwal et al., 2016 [40]
MDA5 protein inhibition by paramyxovirus V proteins Mandhana et al., 2018 [41]
Altering nonstructural proteins of West Nile virus (WNV) affects LC3 modification and aggregation
Martín-Acebes et al., 2015 [43]
2Bacterial infection
Anti-bacterial activity
In Bcl-xL knockout cells, Streptococcus pyogenes infection is promoted Nakajima et al., 2017 [53]
NOD proteins interaction with Atg16L1 and initiation of anti-bacterial autophagosome biogenesis
Sorbara et al., 2013 [212]
Protection from Caenorhabditis elegans infection by transcription factor HLH-30/TFEB-mediated
autophagy Chen et al., 2017 [215]
Inhibition of Mycobacterium tuberculosis in human macrophages by SMAD specific E3 ubiquitin
protein ligase 1 (SMURF1) Franco et al., 2017 [398]
Pro-bacterial activity
Effector Ats-1 is used to enhance autophagosomes formation containing LC3, Beclin 1, Atg8 and
Atg6, without lysosomal marker by Anaplasma phagocytophilum Niu et al., 2012 [222]
Yersinia-containing vacuoles (YCVs) contains autophagy markers but not acidified Moreau et al., 2010 [224]
Coxiella-replicative vacuoles contains LC3, Beclin 1, and Rab24 Vázquez and Colombo, 2010
[226]
Inside BCVs, replication of Brucella requires ULK1, Beclin 1, and Atg14L Starr et al., 2012 [227]
Secreted phospholipases C (PLCs; PlcA and PlcB) and a surface protein (ActA) help Listeria
monocytogenes multiplication Mitchell et al., 2018 [50]
Shigella gatekeeper protein MxiC regulate type III secretion Roehrich et al., 2017 [52]

Cells 2019,8, 674 25 of 64
Table 1. Cont.
S. No. Activity Associated
with Autophagy Effect of Autophagy Modus Operandi of Related Activity and Example/Proof of Concept Reference(s)
3 Tumor
Tumor suppression
Monoallelic loss of Atg6/Beclin 1 gene – correlated with human prostate, breast, and ovarian cancers
Choi et al., 2013 [285]
Beclin 1 overexpression inhibits tumor progression Liang et al., 1999 [286]
Inhibition of necrosis and chronic inflammation through inhibiting- high mobility group box 1
protein (HMGB1) Tang et al., 2010 [288]
Autophagy deficiency- leads to benign hepatoma cell death Takamura et al., 2011 [291]
Autophagy induced by PTEN and TSC, the tumor suppressor protein Feng et al., 2005 [301];
Tsuchihara et al., 2009 [302]
In mice tumor model, inactivation of Beclin 1 and Atg5 affects autophagy Levine, 2007 [292]
Heterozygous disturbance of Beclin 1 lead to development of cancer Qu et al., 2003 [289]; Yue et al.,
2003 [293]
UV radiation resistance associated gene (UVRAG) can suppress tumorigenicity and proliferation of
colon cancer Liang et al., 2006 [295]
Pogostone stimulate autophagy and apoptosis through PI3K/Akt/mTOR axis and have
anti-colorectal tumor activities Cao et al., 2017 [303]
Tumor induction
Autophagy alleviates stressed condition – in hypoxic conditions, metabolic stress, shortage of
energy, damaged mitochondria and other organelles Sato et al., 2007 [306]
Increased autophagy-associated protein LC3 and BNIP3- linked to colorectal and gastric cancers;
Elevated expression of NIP3 (a pro-apoptotic member of the Bcl-2 family of cell death factor) in
gastric carcinomas
Lee et al., 2007a [309]
Autophagy inhibition leads to cell death in tumors acting like an RAS-activated tumor Guo et al., 2011 [312]
In the absence of autophagy -accumulation of ubiquitinylated protein aggregates and higher p62
level- responsible for liver tumor Takamura et al., 2011 [291]
Activation of autophagy and peroxisome proliferator-activated receptor gamma (PPARγ) protect
colon cancer cells against apoptosis Tylichováet al., 2017 [310]
In RAS-activated tumors, inhibition of autophagy leads to increased cancer cell death Guo et al., 2011 [312]
Post-chemotherapy, increased autophagy may cause cancer cells to go into dormancy and
proliferate later White et al., 2010 [315]
Proteasome 26S subunit, non-ATPase 10 (PSMD10) or gankyrin induced autophagy in
hepatocellular carcinoma causes tumor progression Luo et al., 2016 [318]