21. Autophagy 1

complete

(Macro)Autophagy

• What is the definition of autophagy?

• What type of material does autophagy target?

• Why is autophagy called "self-eating"?

• Autophagy, meaning "self-eating," is a mechanism by which cells digest intracellular material.

• Autophagy targets intracellular material, such as damaged organelles, misfolded proteins, and excess cellular components.

• Because it allows cells to digest themselves from the inside, recycling cellular components for energy and repair.

Why do cells need degradation?

Cells need degradation to:

• Maintain homeostasis

• Enable/prevent signalling

• Remove damaged components

• Recycle nutrients

• Facilitate cell reprogramming (e.g., differentiation).

Types of mechanisms of degradation

• What are the two main mechanisms of cellular degradation?

• What is the Ubiquitin-Proteosome System (UPS)?

• How is the proteosome different from autophagy pathways?

• What are the three types of autophagy?
  

• Two main mechanisms:

  • Ubiquitin-Proteasome System (UPS)

  • Autophagy

• The UPS targets proteins for degradation by tagging them with ubiquitin. Proteins are then fed one at a time into the proteosome, a barrel-shaped structure that chops them up.

• The proteosome directly chops up proteins, while all autophagy pathways rely on the lysosome for degradation.

• 3 types of autophagy:

  • Macroautophagy

  • Chaperone-Mediated Autophagy - selective through amino acid target sequences

  • Microautophagy - direct engulfment of cellular material by invaginations in the lysosomal membrane.

Comparisons between degradation pathways

• What are the main characteristics of the proteosome degradation pathway?

• What are the main characteristics of macroautophagy?

• What are the main characteristics of chaperone-mediated autophagy and microautophagy?

• Which degradation pathway can remove whole organelles?

• Which degradation pathway has the highest capacity for bulk digestion?

• Proteosome:

  • Non-lysosomal

  • Degrades individual proteins.

  • Major turnover route for short-lived proteins.

• Macroautophagy:

  • Lysosomal.

  • Bulk digestion pathway capable of removing whole organelles (unique capability).

  • Released molecules can support metabolism.

• Chaperone-mediated autophagy and microautophagy:

  • Both are lysosomal pathways.

  • Degrade individual proteins.

  • Turn over specific, generally long-lived proteins.

  • Relatively low capacity compared to macroautophagy.

• Macroautophagy is the only pathway capable of removing whole organelles.

• Macroautophagy has the highest capacity for bulk digestion, unlike chaperone-mediated or microautophagy.

Functions of (Macro)autophagy: Nutrient recycling

• What is the primary role of macroautophagy in nutrient recycling?

• What happens to cells lacking autophagy during starvation?

• Why are autophagy-deficient mice unable to survive neonatal starvation?

• How does macroautophagy support cancer cells in solid tumors?

• Macroautophagy is rapidly upregulated under starvation to enable non-selective bulk degradation of the cytosol, recycling nutrients to support cell survival.

• Cells lacking autophagy die under starvation because they cannot recycle nutrients effectively.

• Autophagy-deficient mice cannot cope with the sudden lack of maternal nutrient supply after birth, leading to neonatal death.

• Cancer cells in solid tumors rely on macroautophagy to survive under conditions of nutrient deprivation.

Functions of autophagy: Cellular remodelling

• Why is autophagy essential for cellular remodelling?

• What role does autophagy play in erythropoiesis?

• How does autophagy contribute to the removal of sperm-derived mitochondria?

• Autophagy is the only mechanism capable of degrading organelles, which is critical for forming specific cell types during differentiation.

• During erythropoiesis (red blood cell differentiation), autophagy removes unnecessary organelles to create mature red blood cells.

• Autophagy selectively degrades sperm-derived mitochondria after fertilization to prevent paternal mitochondrial inheritance.

Functions of autophagy: Removal of damaged components

• Why is autophagy important for removing damaged components?

• How does autophagy handle mechanical damage to cellular components?

• What is mitophagy, and why is it important?

• What happens if damaged components are not removed by autophagy?

• Autophagy removes damaged cellular components that accumulate over time, maintaining cellular health and preventing dysfunction.

• Autophagy can remove mechanically damaged components, such as those affected during or after exercise.

• Mitophagy is the selective removal of damaged mitochondria via autophagy, preventing the accumulation of dysfunctional mitochondria and reducing oxidative stress.

• If damaged components are not removed, they can accumulate, impair cellular function, and contribute to diseases like neurodegeneration or aging-related disorders.

Functions of autophagy: Removal of damaged components

• How does autophagy contribute to combating cellular damage?

• What happens to lysosomal capacity as we age?

• Why is reduced autophagy a major factor in age-related degeneration?

• Which cell types are most susceptible to reduced autophagy with aging?

• How does reduced autophagy contribute to neurodegenerative diseases?

• Autophagy removes damaged components, which is essential for maintaining cellular health and preventing age-related degeneration.

• Lysosomal capacity decreases with age, reducing the efficiency of autophagy and contributing to the accumulation of cellular damage.

• Reduced autophagy leads to the accumulation of damaged components, impairing cellular function and increasing susceptibility to age-related diseases.

• Long-lived or highly metabolic cells, such as neurons and muscle cells, are most susceptible to reduced autophagy and age-related degeneration.

• Reduced autophagy leads to the accumulation of damaged proteins and organelles in neurons, contributing to diseases like Alzheimer’s, Parkinson’s, and Huntington’s.

Autophagy and living longer

• What is the dietary restriction hypothesis in relation to autophagy?

• What evidence supports the dietary restriction hypothesis?

• What is different about the eat2 mutants of C.elegans?

• How do exercise and starvation impact autophagy?

• How can you increase the lifespan of yeast?

• The dietary restriction hypothesis suggests that starvation or eating less increases autophagy, leading to improved cellular damage repair and increased lifespan.

• Studies on C. elegans showed that dietary restriction extended their lifespan by ~50%. However, this effect was completely lost when autophagy was knocked out.

• eat2 mutants have pharyngeal disfunction, they were used as the C.elegans with restricted diets in the study as they eat less food.

• Both exercise and starvation upregulate autophagy, promoting the repair of cellular damage and improving overall cellular health.

• You can increase the lifespan of yeast by about 3-fold if you grow them in less nutrient rich media.

Functions of autophagy: Killing intracellular pathogens

• How do immune cells like macrophages and neutrophils combat pathogens?

• How do some pathogens evade immune cells?

• What is the role of autophagy in killing intracellular pathogens?

• Why is autophagy crucial for immune defence against pathogens like TB and Salmonella?

• Macrophages and neutrophils engulf bacteria and other harmful agents from the environment to neutralize them.

• Pathogens like TB and Salmonella manipulate phagosome maturation to either replicate within the phagosome or escape into the cytosol.

• Autophagy targets pathogens that escape into the cytoplasm, helping immune cells eliminate them and prevent infection.

• Autophagy provides an additional mechanism to eliminate pathogens that evade traditional phagocytic pathways by escaping into the cytoplasm.

Summary of autophagy in physiology and disease

• How does autophagy help in recycling nutrients?

• How does autophagy contribute to cellular remodeling?

• What role does autophagy play in removing damaged proteins and organelles?

• How does autophagy remove intracellular pathogens?

• Autophagy recycles nutrients to:

  • Support survival during starvation.

  • Provide resources for cancer cells in nutrient-poor environments.

• Autophagy aids in:

  • Erythrocyte differentiation by removing unnecessary organelles.

  • Eliminating sperm-derived mitochondria after fertilization.

• Autophagy clears damaged proteins and organelles, preventing conditions such as:

  • Ageing: Accumulation of damage over time.

  • Muscular dystrophy: Impaired muscle function due to organelle damage.

  • Neurodegeneration: Accumulation of damaged proteins in neurons.

  • Cancer: Prevents initial transformation but may support later growth.

• Autophagy targets and eliminates intracellular pathogens, including:

  • Tuberculosis (TB)

  • MRSA - however MRSA likes to be taken up by autophagosomes and can therefore use that as a pathway to survive

  • Viruses

Cancer & autophagy

• How is autophagy a double-edged sword in cancer?

• How might targeting autophagy benefit solid tumors in cancer?

• Why might stimulating autophagy prevent cancer formation?

• How could stimulating autophagy benefit other diseases?

• What drugs would need developing to modulate autophagy?

Protective Role: Prevents tumour initiation by clearing damaged components.

Supportive Role: Provides nutrients to cancer cells in established tumours.

• Inhibiting autophagy in solid tumours can prevent cancer cells from surviving in nutrient-deprived environments.

• Stimulating autophagy removes cellular damage, reducing the risk of mutations and preventing cancer initiation.

• Stimulating autophagy may help prevent:

  • Ageing: By removing damaged components.

  • Muscular dystrophy: By clearing damaged organelles in muscle cells.

• Drugs:

  • Drugs to stimulate autophagy: Prevent ageing, muscular dystrophy, neurodegeneration, and cancer formation.

  • Drugs to inhibit autophagy: Target cancer cells in nutrient-deprived tumors.Neurodegeneration: By removing toxic protein aggregates.

Discovering autophagy

• How was the lysosome discovered?

• Why was little progress made in understanding autophagy for 30 years?

• What breakthrough showed autophagy occurs in yeast?

• What role did yeast play in advancing autophagy research?

• What genes were discovered?

• The lysosome was discovered using electron microscopy, with autophagy first identified by observing a mitochondrion within a lysosome.

• Progress was limited due to a lack of understanding of the molecular machinery involved in autophagy.

• Scientists found that mutating yeast to have deficient proteases in the vacuole caused vesicles (autophagosomes) to accumulate during starvation, revealing autophagy in action.

• Yeast models allowed researchers to perform genetic screens, identifying 15 autophagy-related genes (Atg genes) critical for autophagosome formation.

• Atg genes

Overview of the autophagy machinery

• What is unique about the formation of autophagosomes?

• What happens after autophagosomes form?

• What are the three main compartments of autophagy machinery?

• What is the role of SNARE proteins in autophagy?

• What is a phagosome?

• Autophagosomes are crescent-shaped, double-membrane vesicles that form by a bilayer curving in on itself—a unique vesicle formation process within the cell.

• Autophagosomes fuse with lysosomes to form hybrid lysosomal zones where their contents are partially or completely digested.

• 3 main compartments:

  • Regulation and initiation: Controlled by the ULK1 complex, which determines how many autophagosomes to make and where to form them.

  • Membrane initiation and expansion: Driven by the PI3K/Vps34 complex, which facilitates membrane formation to capture cytosolic components.

  • Membrane addition and elongation: Involves multiple proteins (e.g lots of Atg proteins) that add membranes to form the autophagosome.

• SNARE proteins mediate the physical fusion of the fully formed autophagosome with the lysosome for content degradation.

• A phagosome is a vesicle with a double membrane that contains cytoplasmic material destined for degradation.

Selective autophagy

• What is selective autophagy?

• How does ubiquitin tagging work in selective autophagy?

• What role do adaptor proteins play in selective autophagy?

• What is a second mechanism for selective autophagy?

• How does autophagy differ during starvation?

• Why is ubiquitin versatile in autophagy?

• Selective autophagy targets specific organelles or proteins for degradation through adaptor proteins and ubiquitin tagging.

• Ubiquitin acts as a small protein tag that attaches to specific proteins or organelles, marking them for degradation.

• Adaptor proteins have:

  1. Ubiquitin-binding domains: Bind to ubiquitinated targets.

  2. Atg-interacting motifs (AIMs): Connect to the autophagy machinery for degradation.

• Some proteins contain direct autophagy-interacting motifs, allowing them to bind directly to autophagosomes without ubiquitination.

• During starvation, autophagy is largely non-selective, degrading bulk cytosol to rapidly generate nutrients for survival.

• Different types of ubiquitin conjugation target specific proteins or organelles, enabling precise control in selective autophagy.