AUTOPHAGY

GROUP 2.11
NANGABI ANNITAH 2020-08-00098
KEBIRUNGI FIONAH 2020-08-00109
BUYAMBI RITAH KARALI 2020-08-00201
MANANA VICTOR 2020-08-00112
BOGERE RUFINAH JEAN 2020-08-00113
LILLIAN NANZALA 2020-08-00255
OJELAM KELLY 2020-04-10716
AUTOPHAGY

•Auto- self Phagy- eating

•Autophagy therefore is the process

by which the cell eats its contents.

• Or autophagy is sequestration/isolation of cellular

organelles into cytoplasmic autophagosomal

membranes that later fuse with lysosomes and the

enclosed materials are digested lysosomal enzymes.

• Autophagy usually occurs in states of nutrient
deprivation and other stress conditions in the cell.
vAutophagy is implicated in either:

1. Physiological states (e.g.., aging and exercise) or

2. Pathological states (cancers, inflammatory bowel
disease, neurodegenerative disorders).

vIt occurs in single-celled organisms

(e.g.bacteria,protists ,yeast) as well as mammalian
cells.
FUNCTIONS OF AUTOPHAGY

• Serves as a survival mechanism under various stress

conditions maintaining the integrity of cells by recycling
essential metabolites and clearing intracellular debris.

• Its involved in the clearance of intracellular aggregates that

accumulate during aging, stress and various disease states.
• It can trigger cell death, if its inadequate to cope with the
stress imposed on the cell.

• Its involved in the turnover of organelles like mitochondria,

lysosomes and endoplasmic reticulum.
Functions of autophagy continued.

• Autophagy can also play crucial roles in adaptive

immune responses such as antigen presentation and
lymphocyte development
• . Autophagosomes can fuse with major-
histocompatibility-complex (MHC) class II loading
compartments therefore facilitates generation of self
tolerant T cells.
 Classification of autophagy
Autophagy is classified depending on mechanism
of delivery of the cellular contents to the lysosome
and this is as follows;
1. Chaperone mediated autophagy; cellular
contents are carried to the lysosome by
chaperone proteins e.g.. Heat Shock cognate
70( HSC70).
1. Micro autophagy; Involves inward invagination of
the lysosome membrane for delivery of contents.
2. Macro autophagy; Involves the a double membrane
bound autophagic vacuole fusing with a lysosome.
 Chaperone mediated autophagy

• This involves the direct translocation of the

cellular contents across the lysosomal
membrane by use of chaperone proteins such
as Heat shock cognate 70(HSC70).
• The proteins to be degraded have a specific target
motif in their amino acid sequence (KFERQ like-motif
bound substrate) which binds to the cytosolic
chaperone HSC70.
• Q- Glutamine ,R-Basic amino acids, E-acidic amino
acids, F-bulky amino acids, K-basic or bulky amino
acids.

• The chaperone-substrate complex then binds to

cytosolic tail of lysosomal associated membrane
protein type 2(LAMP2A) leading to unfolding of
the substrate.
• The substrate is then translocated into the lysosome
followed by its subsequent degradation by lysosomal
enzymes
 Micro autophagy

Ø This is a non selective lysosomal

degradation process that involves
invagination and engulfment of cytosolic
cargo at the boundary membrane of the
lysosome.
 Macro autophagy

Ø Macro autophagy referred to as

autophagy, the major form of autophagy
involving the sequestration and
transportation of portions of cytosol in a
double membrane bound autophagosome.
Steps of autophagy
• Formation of an isolation membrane also known as the
phagophore
• its nucleation;
ü the isolation membrane is believed to be derived from the
ER, though plasma membrane or mitochondria can also
contribute.
• Elongation of the phagophore inside which intracellular
organelles and cytosolic structures are sequestered.
• Maturation of the autophagosome,
• fusion with lysosomes,and
• degradation of the contents
ILLUSTRASTION.
Components of the initiation complex.

• ULK 1 &2( Uncoordinated 51-like kinase 1&2)

• FIP 200 (Focal adhesion kinase family interacting
proteins)
• ATG 13
• ATG 101
Components of the nucleation complex.
• VPS 34 (Vacuolar protein sorting 34)
• Beclin 1
• BCL-2 family proteins
• UVRAG ( ultra violet radiation resistance associated gene
11q13.5)
• VPS15
• P115
• ATG14L
 INITIATION.

• Environmental cues like starvation or depletion of growth

factors activate an initiation complex of four proteins that
stimulates the assembly of a nucleation complex.

üThe proteins of the initiation complex are encoded by

autophagy related genes
• AMPK and mTOR are highly conserved kinases which
sense energy and nutrient stress and so influence
Atg1 activity to regulate autophagy.
• In response to growth factors that stimulate the class I
phosphatidylinositol 3-kinase–AKT pathway, or other
nutrient related signals (e.g.., leucine), mTORC1
negatively regulates a complex consisting of UNC-
51–like kinase 1 (ULK1), ATG13, ATG101, and FIP200.
• Upon withdrawal of growth factors ,AMPK is activated
and inhibits mTOR by phosphorylation of TSC2 at( Ser-
1345, Thr-1227) and Raptor at (Ser- 792, Ser-722)

• Inhibition of mTORC1 permits activation of

ULK1(Atg1) — a crucial initiating step in autophagy
• On activation, the initiation complex causes pinching
off of part the ER to form the phagophore.
NUCLEATION.

ØNucleation is the process of mobilizing a small group of

molecules to the point of assembly.
• The Beclin 1–interacting complex, a regulatory platform,
consists of Beclin 1, BCL-2 family proteins (which inhibit
autophagy), the class III phosphatidylinositol 3-kinase
(VPS34), and ATG14L (required for autophagy).
• Stimulation of this complex generates
phosphatidylinositol-3-phosphate, which promotes
autophagosomal membrane nucleation.
ELONGATION
• The autophagosomal membrane elongates further, surrounds and captures its cytosolic cargo,
and closes to form the autophagosome.

• Autophagosomal elongation requires two ubiquitin-like conjugation systems.

i. ATG12 and ATG5 conjugation system.

• Ubiquitin-like protein ATG12 is activated by ATG7 (E1-like) enzyme and then conjugated to
ATG5 by ATG10 (E2-like) enzyme.

.
• The resulting ATG5–ATG12 forms a complex with ATG16L1, which
participates in elongation of the autophagic membrane.

• The ATG12-ATG5-ATG16L1 complex that is formed acts as E3

enzyme in ubiquitination.
ii A second conjugation system requires the ubiquitin-like
protein microtubule–associated protein 1 light chain 3 (LC3,
ATG8).

• LC3 and its homologues are modified with the cellular lipid
phosphatidylethanolamine (PE).
• The precursor form of LC3 is cleaved by the protease ATG4B to generate
the LC3-I form, with an exposed lipid conjugation site at the C-terminal
glycine residue.

• Activation of LC3-I occurs from the sequential action of ATG7 (E1-like)

enzyme and and conjugation of PE to LC3-I is by the action of ATG3 (E2-
like) activities to form LC3-II.
• LC3II interacts by adding phosphotidylethanolamine
to growing phagophore to enable elongation.

NOTE; PE lapidated LC3 is increased during autophagy

and therefore is a useful marker of cells in which
autophagy is occurring.
MATURATION
ØMaturation refers to the complete closure of the
elongated phagophore to form the autophagosome.
• The maturation is mediated by CHMP2A.
• During autophagy, CHMP2A tanslocates to the phagophore
and regulates the separation of the inner and outer
autophagosomal membranes to form the double
membrane autophagosome.
• It should be noted that the mechanism of closure is
however not clear.
FUSION WITH A LYSOSOME

• The newly formed autophagosome fuses with endosomes

and then finally with lysosomes OR directly with the
lysosome to form an autophagolysosome.

• Fusion is mediated by the SNARE(soluble N ethyl amide

sensitive factor attachment protein receptor) proteins.
These include;
üsynaptobrevin,
üSNAP(Soluble NSF attachment protein)-25
üSyntaxin,
DEGREDATION
• In the terminal step, the inner membrane and enclosed
cytosolic cargoes are degraded by lysosomal enzymes.

• There is some evidence that autophagy is not a

random process that engulfs cytosolic contents
indiscriminately.
Stages Cont..

• Instead, it appears that the loading of

cargo into the autophagosome is “selective”
and that one of the functions of the LC3
system is to “target” protein aggregates
and effete organelles.
Factors for selection of proteins as cargo.
• P62/SQSTM1/Ref(2) P
• Nbr1
• Parkin
• Nix
 ROLE OF AUTOPHAGY IN DISEASE
• Cancer ; autophagy may promote growth of cancer cells or
act as a defense against cancer.

• Neurodegenerative disorders; these are associated with

dysregulation of autophagy. In AD and HD, there’s
accumulation of proteins yet autophagy is impaired and
accumulation of toxic substances
• Infectious diseases; microbial proteins of the infectious agents

are degraded via autophagy and delivered for antigen

presentation.

• Inflammatory bowel diseases like Crohn disease and

ulcerative colitis have been linked to SNPs in Autophagy

related genes.
 ROLE OF AUTOPHAGY IN:
CANCER.
• Autophagy plays major roles in the maintenance of
cellular homeostasis.

• Autophagy plays a dual role in cancer.

a)Tumor suppression.
b)Tumor promotion
Tumor suppression.
• Autophagy operates as a mechanism for tumor suppression via
reduction of damaged cellular parts and proteins and this can further
be explained in the following ways;

i. Autophagy prevents tumor generation through the regulation of

reactive oxygen species by degrading damaged mitochondria.
• Autophagic degradation of the immune checkpoint
protein PD-L1(Programmed Death Ligand 1) generally
enhances T cell activation and suppresses tumor
growth.
• In addition, autophagy can modulate the immune response
against cancer cells by promoting the presentation of tumor
antigens to immune cells, thereby enhancing the antitumor
immune response.

• Some drugs like Afatinab are autophagy modulators tat are

used in cancer treatment. Afatinab acts by suppressing
mTORC1 which initiates autophagy in cancer cells
Tumor promotion
• Several studies indicate that autophagy acts to promote tumor
survival and growth in advanced cancers.

• Tumors are exposed to extremely stressful conditions, including

hypoxia and nutrient deprivation and autophagy can help the cells to
overcome these stresses.
• Under hypoxia conditions, hypoxia-inducible factor-1
alpha (HIF-1) induces activation of autophagy in
cancer progression hence causing recycling of cellular
constituents to form energy.
• Autophagy fulfills the high metabolic and energetic demands
of proliferating tumors by recycling intracellular components
to supply metabolic substrates

• In addition, autophagy can contribute to tumor progression by

supporting cancer cell invasion and metastasis. Autophagy can
promote the secretion of proteases and other factors that
degrade the extracellular matrix, allowing cancer cells to
migrate and invade neighboring tissues.
IMPORTANT GENES MUTATED IN AUTOPHAGY

1.UVRAG (Ultraviolet radiation resistance associated gene).

11q13.5.

2.BECN 1 gene a tumor suppressor encodes for Beclin 1.

Mutation in BECN1 causes absence or impaired Beclin1 hence
no formation of phagophore. 17q21.31.
 ROLE IN NEURODEGENERATIVE DISEASES.

• The neurodegenerative diseases are age-dependent

hereditary or sporadic disorders that are manifested by
progressive loss of neural function.
• Common features in their pathogenesis are mitochondrial
dysfunction and the accumulation of protein aggregates as a
result of mutation and impaired clearance mechanisms.
• Autophagy is dysregulated in neurodegenerative disorders.

• For example, in the brains of persons Alzheimer’s disease, the

accumulation of autophagosomes is accelerated.
• Mutations in genes like ATP6AP2 and Presenilin 1 that
regulate lysosomal acidification are associated with X-
linked Parkinson's and Alzheimer's disease due to
impaired vATPase assembly resulting in autophagy
defects.
• Alzheimer's disease involves aberrant accumulation of
hyperphosphorylated forms of microtubule-associated protein
tau leading to the formation of neurofibrillary tangles and the
accumulation of beta amyloid peptide (AB) in neural plaques.

• AB may impair lysosomal function and autophagic clearance of

this protein.
• Furthermore, the autophagosome, which contains
gamma-secretase and related enzymes involved in the
generation of AB from precursor forms, may provide a
source of AB under conditions of impaired
autophagosome-lysosome fusion.
 ROLE IN INFECTIOUS DISEASES;
• Autophagy contributes to the regulation and function of innate and adaptive
immune responses.

•
• Several medically important human pathogens are degraded in vitro
by xenophagy, including bacteria (e.g.., group A streptococcus,
Mycobacterium tuberculosis, Shigella flexneri, Salmonella enterica,
Listeria monocytogenes, and Francisella tularensis), viruses such as
herpes simplex virus type 1 (HSV-1) and chikungunya virus, and
parasites such as Toxoplasma gondii.
• Autophagy genes have been shown to play a protective role in vivo against
the above pathogens.

• Macrophage specific deletion of Atg 5 increases susceptibility to

tuberculosis.

• Autophagy as an immune mechanism controls inflammation and acts as a

cell-autonomous defense against intracellular microbes.
 ROLE IN INFLAMMATORY DISEASES.

Crohns disease.
• A chronic inflammatory bowel disease characterised by patchy deep
ulcers.

• Patients with Crohn’s disease have an over abundance of a type of

gut bacteria called adherent-invasive Escherichia coli (AIEC), which
promotes inflammation in the intestine
ØNOD2, located on chromosome 16q12.21, was the first disease-
susceptibility gene discovered for CD.

ØNOD2(Nucleotide binding oligomerisation containing protein2) is involved

in:

• intracellular bacterial sensing

• Autophagosome formation

• Recruitment of ATG16L1 located on 2p37.1 .

ØATG16L1 is important in:

• Autophagosomal formation

• Suppressing paneth cells.

• Mutant NOD2 fails to recruit ATG16L1 to the plasma membrane and
wrapping of invading bacteria by autophagosomes impaired.
• When the mechanism of autophagy is impaired, lipopolysaccharides
and damage-associated molecular patterns(DAMPs) trigger signaling
by stimulating TLR and NOD-like receptors, tumor necrosis factor
(TNF), and other inflammatory cytokines causing damage to the
tissue.
ROLE IN CARDIOVASCULAR DISEASES

• Mitochondria damaged by various stresses( ischaemia, hypoxia,

oxidative stress, and metabolic dysregulation) are degraded by
autophagy.
• When mtDNA is not completely digested during the mitophagic
processes, it is recognised by TLR9 which activates inflammatory
responses.

• Inflammatory responses induced by damaged mitochondrial DNA

(mtDNA) in the heart leads to heart failure.
• An X-linked mutant of lysosome-associated membrane protein 2
(LAMP 2) causes Danon disease.

• LAMP2 deficiency disrupts the fusion between autophagosome

and lysosome, leading to decreased autophagy flux and increased
accumulation of autophagic vacuoles.
Role of autophagy in Aging
• The homeostatic functions of autophagy with respect to turnover of long-lived
proteins and removal of damaged organelles and cellular debris are believed
to constitute an antiaging process.

• Aging is similar to neurodegeneration in that the accumulation of unprocessed

material accelerates while the function of compensatory mechanisms (i.e.,
autophagy) declines.
• Analysis of gene expression in the brain in old persons
as compared with young persons revealed down-
regulation of autophagy genes (ATG5, ATG7, and
BECN1) with age and, conversely, up-regulation in
persons with Alzheimer’s disease.
 ROLE OF AUTOPHAGY IN
FASTING

Fasting ;is purposeful restraining from eating and sometimes

drinking .
Starvation ;is severe deficiency in energy and calories intake below
the level necessary to maintain a living .
 TYPES OF FASTING
• Dry fasting; is a form of intermittent fasting in which no liquid is
consumed during the fasting window .

• Wet fasting; is the type where no drink or food is consumed

besides water.

• Intermittent fasting; fasting consisting of various meals timing

schedules that cycle between voluntarily fasting and no fasting for
a period of time .
 forms of intermittent fasting.
• Alternative day fasting ,it involves alteration between a 24 hour
fasting day when a person eats less than 25% usual energy
needs .

• Periodic fasting ,involves any of the consecutive fasting of more

than 24hours .

• Time restricted fasting, Eating only during certain hours e.g.

16:8 hour diet .
 Benefits of fasting .
• Reduced inflammation and oxidative stress symptoms
• Lipolysis activity
• Modulated hormone activity e.g. leptin and ghrelin
• Aids in weight loss
• Reduces Aging
• Induces autophagy
 Mechanism
• AMPK is a highly conserved heterotrimeric Ser/Thr Kinase that
monitors cellular energy status through in the AMP/ATP and ADP/ATP
ratios

• Under conditions of falling energy status ,AMPK promotes ATP

production by regulating the activity or expression of catabolic
proteins and conserves ATP levels by down regulating biosynthetic
pathways.
• Metabolically, AMPK activation during energetic
challenge Promotes the transition from glucose to
lipids as the main substrate for cellular oxidation.
• Serine -threonine kinase AKT(protein kinase B)mediates the
suppression of carbohydrate metabolism in favor of lipid
oxidation during fasting.

• Because AKt promotes anabolic and lipogenic pathways

and mediates the effect of insulin on cellular glucose uptake,
its activity would be expected to fall when animals are
fasting .
References
• Robbins and Cotran, Pathologic Basis of Disease, Ninth
Edition.
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6274
804/#:~:text=Autophagy%20inhibits%20necrosis%20a
nd%20inflammation,important%20factor%20in%20ca
ncer%20metastasis.
• Mechanisms of Disease; Review article Autophagy in
Human Health and Disease; Augustine M.K. Choi, M.D.,
Stefan W. Ryter, Ph.D., and Beth Levine, M.D
CORRECTIONS
• What are endosomes?
• Endosomes are membrane-bound organelles found in
eukaryotic cells that function in the sorting, processing,
and transport of molecules between different
compartments of the cell.
• What is invagination?
• Invagination is a biological process in which a portion of a
cell membrane or tissue folds inward and creates a pocket.
How do the different types of autophagy differ?

Chaperone mediated Macro autophagy Micro autophagy

autophagy
Involves direct Chaperone
How do the Macro-autophagy
mediated
different Macro
typesautophagy Micro-autophagy
of autophagy Micro autophagy
differ?
autophagy
transportation of Involves direct
involves the Macro-autophagy
formation involves the direct involves
Micro-autophagy
protein aggregatestransportation
into of aof double-
protein involves the formationengulfment
of the directof
engulfment of
aggregates into a double-membraned cytoplasmic material by
lysosomes for membraned
lysosomes for
vesicle
vesicle called an
cytoplasmic material by
the lysosome through an
degradation. degradation.called an autophagosome around the lysosome
invaginationthrough
of the
the cytoplasmic material lysosomal membrane.
autophagosome around an invagination of the
to be degraded.
the cytoplasmic lysosomal membrane.
This type ofmaterial
autophagy isto be degraded.
Macro autophagy is also Micro autophagy is non
selective. selective selective.

This type of autophagy Macro autophagy is also Micro autophagy is non

is selective. selective selective.
What are the benefits of fasting?

• 1. Reduces inflammation
• This is through induction of autophagy which removes damaged cells
there by preventing signaling of inflammatory cells thus reducing
inflammation.
• Autophagy also removes microorganisms which induce inflammation.
• 2. Reduces Aging
• Fasting induces autophagy which removes the accumulated proteins
and damaged organelles there by enhancing anti-aging mechanisms.
• 3. Lipolysis activity
• During fasting the glucose stores are depleted, so the body resorts to
breakdown of lipids for cellular oxidation to release ATP.
Benefits of fasting contd.

• 4. Aids in weight loss

• Fasting leads to weight loss through lipolysis.
• It also affects ghrelin hormone by decreasing it which decreases the
hunger as well thus reducing the number of calories that are
consumed and resulting into loss of weight.
• 5. Modulation of hormones e.g. ghrelin and leptin.
• During fasting, there is activation of the leptin hormone which is an
appetite suppressant and increases fat metabolism therefore will be
an increase in its levels and there is also a decrease in the levels of
ghrelin which is a hunger hormone.
•
• When does autophagy fight cancer?
• In the early stages
•
• What is a phagophore?
• Phagophore is a cap shaped active sequestering membrane of autophagy.
• Outline the steps of autophagy.
• Initiation
• Nucleation
• Elongation
• Maturation
• Fusion
• Degradation
• The ubiquitin like conjugating enzyme
• i) In the ATG12-ATG5 conjugation system.
• Atg7 (E1 like); activation of ATG12
• ATG10 (E2 like); conjugation of ATG12 to ATG5
• ATG12-ATG5-ATG16 complex (E3 like); stimulates LC3-II to transfer
phosphotidylethanolamine to the elongating phagophore.
•
• ii) In the LC3, ATG8 conjugation system
• ATG7 (E1 like); for activation of LC3-I
• ATG3 (E2 like); for conjugation of PE to LC3-I
• LC3-II/PE-LC3-I complex (E3 like)
• By what mechanism does the CHMP2A lead to phagophore closure
to form an autophagosome?
•
• CHMP2A- Charged Multivesicular body Protein 2A
• ESCRT- Endosomal Sorting Complexes Required for Transport.
• This protein exists as a compartment of ESCRT-III as a regulator of
phagophore closure.
• During autophagy, CHMP2A tanslocates to the phagophore and
regulates the separation of the inner and outer autophagosomal
membranes to form the double membrane autophagosome.
•