Lecture 7 Flashcards
(11 cards)
What are lysosomes?
Lysosomes are acidic organelles with ≈ 63 hydrolases that degrade, DNA, RNA, protein, lipid and carbohydrate
Lysosomes receive material from the endocytic, phagocytic and autophagy pathways
Lysosomes allow nutrients to be recycled
Routes to the lysosome – receptor mediated endocytosis, fluid phase
What is autophagy?
Autophagy is a process of self-cannibalisation.
Cells capture their own cytoplasm and organelles and deliver them to the lysosome for nutrient release.
Selective autophagy often captures ubiquitinated cargo
Pre- autophagosomal structures nucleate to form phagophore
Surround what is going to be degraded
Delivered to lysosome
Autophagosome fuses with lysosome and forms autolysosome, material digested and lysosome recycled
Peroxisome and mitochondrion surrounded by double membrane – autophagosome
Describe how Autophagy Regulates Energy Homeostasis
Initiators include: Starvation, Ammonia stress, Damage, Developmental cues
Traditionally autophagy is known as a starvation response, whereby material is degraded to the building blocks to regulate energy.
Starvation triggers autophagy
Lack of nutrients sends stuff to lysosome by autophagy
Proteins broken down into amino acids
Describe the relationship between autophagy and disease
The consequences of impaired autophagy include, lack of nutrients, aggregate formation, immune activation, ROS accumulation and even chronic infection
Lots of cargo ubiquitinated, binds to p62 which binds to LC3
Damaged, aged mitochondria are enlarged, release ROS, impairs autophagy and leads to oxidative damage, sign of ageing
Reduce ageing – reduce amount of damaged mitochondria
Explain Balance between Beneficial and Detrimental Factors During Ageing
Lysosomal function is crucial for clearing damaged molecules/organelles
A non-functional or aged lysosome which is not as effective leads to protein aggregates, damaged mitochondria etc. -> ageing
Balance between beneficial and detrimental factors during aging. Declining levels or activities of beneficial factors and accumulation of detrimental factors during aging lead to fitness collapse at the cellular level. The scheme enumerates several beneficial and detrimental factors during lifespan. Beneficial factors include rejuvenation molecules such as TIMP2 , damage clearance mechanisms and organelle functions, whereas detrimental factors include DNA damage, protein aggregates and the micro-environment created by senescent cells.. For instance, mitochondrial functions decline, including the accumulation of mutations in mitochondrial DNA. In addition, the lysosomal pH increases with a resulting decrease in its degradation capacity because hydrolases work at acidic pH (dark gray circles in aged cell). Furthermore, there is also a loss of nuclear integrity and genome stability, as well as a shortening of telomeres and an accumulation of protein aggregates
What is Transcription Factor EB (TFEB)?
Microphthalmia-transcription factor E (MiT/TFE) subfamily of basic helix-loop-helix transcription factors bind similar CLEAR sequences
TFEB overexpression in HeLa cells upregulated lysosomal gene expression
TFEB regulates lysosomal/autophagy gene expression
Describe how TFEB is regulated by phosphorylation including the mammalian target of Rapamycin complex 1 (mTORC1)
mTORC1 is a rapamycin-sensitive multi-subunit protein complex that contains the PI3K-related kinase mTOR
Active mTor is associated primarily with the lysosome membrane
In the presence of nutrients, mTOR is active and associated with the lysosome. It is a serine/threonine kinase - phosphorylates TFEB which inactivates it. TFEB then binds the chaperone 14-3-3 – doesn’t go to the nucleus, you don’t get any transcription. When there is stress/lack of nutrients mTOR is released from the lysosome surface, it becomes inactive, TFEB can be dephosphorylated by the phosphatase calcineurin where it can then translocate to the nucleus and induce lysosomal/autophagic gene expression.
Explain how mTORC1 Activation on the Lysosome is related to the nutritional status of the Cell
Absence of amino acids Rag GTPases are inactive
mTORC1 is in cytoplasm (inactive)
In the presence of amino acids Rag GTPases are active
Rag recruits mTORC1
Insulin dissociates tuberous sclerosis complex (TSC)
Rheb binds GTP and activates mTORC1
Following on from previous slide active mTOR phosphorylates TFEB inactivating it, reducing lysosomal and autophagy gene expression
The take home message here is that there is a variety of complexes on the extracellular side of the lysosomal limiting membrane which signals to the cell about the nutritional status. Plenty of ‘food’ mTOR is located to the surface of the lysosome where it in activated. Under stress or starvation mTORC1 complex is cytoplasmic and therefore inactive.
Explain how mTOR is a key kinase at the lysosomal membrane regulating autophagy
mTOR phosphorylates ULK1 and ATG13 in the presence of growth factors and nutrients. Thereby preventing phagophore formation. Under starvation/stress mTOR is not activated and furthermore AMPK will phosphorylate mTOR inhibiting it. Inhibiting mTOR leads to dephosphorylation of ATG13 and ULK1 leading to PAS formation and subsequent autophagosome formation.
Explain how mTORC1 Regulates Autophagy AND Autophagy/lysosomal gene transcription
mTOR released from lysosome and inactive
TFEB is not-phosphorylated - lysosome/autophagy gene transcription is upregulated
ULK1 complex is not phosphorylated and autophagy is induced
mTOR lysosome associated and active
TFEB is phosphorylated - lysosome/autophagy gene transcription reduced
ULK1 complex is phosphorylated and autophagy is inhibited
Explain the mitochondrial-lysosomal axis theory of Ageing
‘Old’ mitochondria create more ROS and eventually oxidative stress
H2O2 enters the lysosome and redox-active iron creates hydroxyl radicals which damages macromolecules leading to an accumulation of lipofuscin (which is undigestible)
Undigestible lipofuscin leads to a loss of lysosomal activity i.e. LYSOSOMES AGE!
