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AutophagyPart 1

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• Introduction• Types of autophagy• Cellular and Molecular mechanisma• Signaling pathways• Estimation of Autophagic Activity

Autophagy

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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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• 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.

Introduction

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Types of autophagy

• Macro-autophagy• Micro-autophagy• Chaperone-mediated autophagy,

General feature for all of them : proteolytic degradation of cytosolic components at the lysosome.

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Micro-autophagy

• By Invagination of the lysosome membrane, cytosolic components are directly taken up by the lysosome itself through.

• It could be selective or non-selective

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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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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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The basic machinery of autophagy

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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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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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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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Molecular Mechanism

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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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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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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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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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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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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 carboxyterminalglycine 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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• 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.

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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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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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