Autophagy is a critical cellular process that involves the lysosomal degradation of cytoplasmic components, crucial for maintaining homeostasis and protecting against various diseases. It encompasses several types, primarily macroautophagy, and involves a complex molecular machinery including autophagy-related genes (atg) that regulate its initiation, elongation, and degradation stages. Key mechanisms include the formation of phagophores, the processing of LC3 proteins, and the fusion of autophagosomes with lysosomes for effective cellular recycling.

1. Autophagy
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2. Autophagy
• Introduction
• Types of autophagy
• Cellular and Molecular mechanism
• Signaling pathways
• Estimation of Autophagic Activity
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3. Introduction
• Auto-phagy = Self- eating
• Autophagy is a lysosomal degradation pathway that is essential for
survival, differentiation, development, and homeostasis.
• Autophagy principally to protect organisms against diverse pathologies,
including infections, cancer, neurodegeneration, aging, and heart
disease.
• First autophagy in rat liver cells
• But it was not clear why? and how it works?
• Starvation, Hypoxia, Stress…
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4. Introduction
• How autophagy is regulated and executed at the
molecular level have been made in yeast.
• 32 different autophagy-related genes (Atg)
• many of these genes are conserved in slime
mould, plants, worms, flies and mammals.
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5. Types of autophagy
General feature for all of them : proteolytic degradation of
cytosolic components at the lysosome.
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6. Micro-autophagy

7. Chaperone-mediated autophagy
(CMA)
• Targeted proteins are translocated across
the lysosomal membrane in a complex
with chaperone proteins (such as Hsc-70)
that are recognized by the lysosomal
membrane receptor lysosomal-associated
membrane protein 2A (LAMP-2A),
resulting in their unfolding and
degradation.
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8. Macro-autophagy
• Delivers cytoplasmic cargo to the lysosome
through autophagosome
(a double membrane-bound vesicle)
• Autophagosome fuses with the lysosome
to form an autolysosome.
• It could be selective or non-selective
• The most important type is
macroautophagy, referred to as
‘autophagy.
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11. The basic
machinery of
autophagy
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12. Initiation
• Start from a phagophore:
lipid bilayer isolated
membrane
• Contributed by the
endoplasmic reticulum
(ER) and/or the trans-Golgi
and endosomes
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13. Elongation
• This phagophore expands to engulf intra-cellular cargo,
such as protein aggregates, organelles and ribosomes,
thereby sequestering the cargo in a double-membraned
autophagosome.
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14. Maturation- Degradation
The autophagosome matures and fuses with the
lysosome, promoting the degradation of autophagosomal
contents by lysosomal acid proteases.
Re-use
So is autophagy
‘recycling factory’ ?
- ATP generation
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15. Molecular
Mechanism
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16. Molecular stages
• Phagophore formation or nucleation
• Atg5 –Atg12 conjugation, interaction with Atg16L and multimerization at the
phagophore.
• LC3 processing and insertion into the extending phagophore membrane.
• Capture of random or selective targets for degradation.
• Fusion of the autophagosome with the lysosome, followed by proteolytic
degradation by lyso-somal proteases of engulfed molecules.
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18. 1- Phagophore formation
(nucleation)
• In mammalian cells, phagophore
membranes initiate from the ER,
Golgi and even nucleus.
• Atg1 kinase (ULK1) with Atg13 and
Atg17 regulate Atg9 activity which
promotes lipid recruitment to the
expanding phagophore .
• TOR Kinase prevent Atg 13 to
interact Atg 1
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19. Phagophore
formation
(nucleation)
• (vesicular protein sorting 34) from class III PI-3 kinases
Using phosphatidylinositol (PI) to give (PI3P), for elongation .
• When it binds to Atg6/Beclin-1 it will be more active.
• Beclin-1 is mono-allelically deleted in human breast,
ovarian and prostate cancer.
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20. Phagophore formation (nucleation)
• We still need to know how these regulatory proteins move to
attach the ER. ( Ambra…..)
• But what we know is Bcl-2 and Bcl-XL are able to disrupt the
interaction between Beclin1 and VPA34.
• Starvation JNK1 to phosphorylate Bcl-2
• So what is the function of Bcl-2 protein ?
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23. 2- Atg5 –
Atg12
conjugation
• Atg7 activates Atg12 and transfers
it to Atg10 .
• Atg10 helps to bind Atg 12 to Atg5
then to Atg16L .
• The complex induces curvature
into the growing phagophore
through asymmetric recruitment
of pro-cessed LC3B-II
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24. 3- LC3
processing
• Atg8/ LC3B is a cytosolic protein that,
upon induction of autophagy, is
proteolytically cleaved by Atg4 (Atg4, a
cysteine protease) to give LC3B I
• More activation of LC3B I by Atg7 (
adding carboxyterminal
glycine residue)
• LC3B I transferred to Atg3( Carrier) and
adding of
phos-phatidylethanolamine (PE) to
generate LC3B-II.
(Atg5 – Atg12) locates LC3BII in the into the
growing phagophore
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26. • The synthesis and
processing of LC3 is
increased during
autophagy, making it a
strong marker.
• Once it is in, it acts on
hemifusion of membranes
and in selecting cargo for
degradation.
Dr Aliwaini 26

27. 4- Capture of
random or
selective
targets for
degradation.
• LC3B-II, acting as a ‘receptor’ to interacts
with ‘adaptor’ molecules as p62/SQSTM1
and NBR1on the target to promote their
selective uptake and degradation.
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28. 5-
Autophagosome
fusion to
lysosome to
form the
‘autolysosome’
• The cytoskeleton also plays a role in
autolysosome formation , a microtubule
poisons nocadazole prevent diffusion.
Once fused:
1- cathepsin proteases B and D
2- Lamp-1 and Lamp-2
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