Autophagy Part 1 Dr Aliwaini 1 Autophagy Introduction

Autophagy Part 1 Dr Aliwaini 1

Autophagy • • • Introduction Types of autophagy Cellular and Molecular mechanisma Signaling pathways Estimation of Autophagic Activity Dr Aliwaini 2

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… Dr Aliwaini 3

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. Dr Aliwaini 4

Types of autophagy • Macro-autophagy • Micro-autophagy • Chaperone-mediated autophagy, General feature for all of them : proteolytic degradation of cytosolic components at the lysosome. Dr Aliwaini 5

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 Dr Aliwaini 6

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 2 A (LAMP-2 A), resulting in their unfolding and degradation. Dr Aliwaini 7

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. Dr Aliwaini 8

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The basic machinery of autophagy Dr Aliwaini 10

Initiation • Start from a phagophore: lipid bilayer isolated membrane • Contributed by the endoplasmic reticulum (ER) and/or the trans-Golgi and endosomes Dr Aliwaini 11

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. Dr Aliwaini 12

Maturation- Degradation The autophagosome matures and fuses with the lysosome, promoting the degradation of autophagosomal contents by lysosomal acid proteases. So is autophagy ‘recycling factory’ ? - ATP generation Re-use Dr Aliwaini 13

Molecular Mechanism Dr Aliwaini 14

Molecular stages • Phagophore formation or nucleation • Atg 5 –Atg 12 conjugation, interaction with Atg 16 L and multimerization at the phagophore. • LC 3 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. Dr Aliwaini 15

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1 - Phagophore formation (nucleation) • In mammalian cells, phagophore membranes initiate from the ER, Golgi and even nucleus. • Atg 1 kinase (ULK 1) with Atg 13 and Atg 17 regulate Atg 9 activity which promotes lipid recruitment to the expanding phagophore. • TOR Kinase prevent Atg 13 to interact Atg 1 Dr Aliwaini 17

Phagophore formation (nucleation) • (vesicular protein sorting 34) from class III PI-3 kinases Using phosphatidylinositol (PI) to give (PI 3 P), for elongation. • When it binds to Atg 6/Beclin-1 it will be more active. • Beclin-1 is mono-allelically deleted in human breast, ovarian and prostate cancer. Dr Aliwaini 18

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 Beclin 1 and VPA 34. • Starvation JNK 1 to phosphorylate Bcl-2 • So what is the function of Bcl-2 protein ? Dr Aliwaini 19

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2 - Atg 5 – Atg 12 conjugation • Atg 7 activates Atg 12 and transfers it to Atg 10. • Atg 10 helps to bind Atg 12 to Atg 5 then to Atg 16 L. • The complex induces curvature into the growing phagophore through asymmetric recruitment of pro-cessed LC 3 B-II Dr Aliwaini 22

3 - LC 3 processing • Atg 8/ LC 3 B is a cytosolic protein that, upon induction of autophagy, is proteolytically cleaved by Atg 4 (Atg 4, a cysteine protease) to give LC 3 B I • More activation of LC 3 B I by Atg 7 ( adding carboxyterminal glycine residue) • LC 3 B I transferred to Atg 3( Carrier) and adding of phos-phatidylethanolamine (PE) to generate LC 3 B-II. (Atg 5 – Atg 12) locates LC 3 BII in the into the growing phagophore Dr Aliwaini 23

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• The synthesis and processing of LC 3 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 25

4 - Capture of random or selective targets for degradation. • LC 3 B-II, acting as a ‘receptor’ to interacts with ‘adaptor’ molecules as p 62/SQSTM 1 and NBR 1 on the target to promote their selective uptake and degradation. Dr Aliwaini 26

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 Dr Aliwaini 27

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