Lecture 14 autophagy Flashcards
What are the steps in the IL2 signaling pathway?
- IL-R is activated upon binding IL-1. Myd88 is recruited to the receptor, facilitating downstream signaling.
- MyD88 recruits IRAK1 (interleukin associated kinase 1) and there is oligomerisation and activation of TRAF6 (TNF receptor-associated factor 6). TRAF6 undergoes ubiquitination specifically K63-linked polyub. Acts as a scaffold to recruit TAK1 (TGF-beta activated kinase 1) complex.
- Activation of the TAK1 in complex with TAB2 and TAB3 phosphorylates IKK complex. This consists of IKKalpha, beta and NEMO (NF0kB essential modulator). IKK activates NF-kB signaling by phosphorylation of IkBalpha. 4. IkBalpha is ubiquitinated by K48 polyub and targeted for proteosomal degradation. This releases NF-kB TF which is composed of p65 and p50 subunits.
- NF-kB translocates to the nucleus. Binds to NF-kB target gene promoters, initiating transcription of genes involved in inflammation, immune response and cell survival.
What is a negative regulator of NF-kB?
CYLD and A20 acts as deubiquitinates (DUBs) that remove K63-linked polyub chains from TRAF6 and other components.
What is an example of a branched ubiquitin chain?
Lysine63 and lysine 48 linked branch chain for the ubiquitination of IKBalpha in the IL2 signaling.
What does HUWE1 do?
E3 ligase that can build a lysine 48 chain off the lysin 63 and act as a blocker to the CYLD. A unique ubiquitin coding signal that specifically affects recognition by downstream reader proteins to enhance NF-kB signaling. CYLD is an inhibitor of ubiquitination of TRAF6.
What is chaperone mediated autophagy?
Soluble cytosolic proteins containing a degenerate sequence (related to KFERQ) are degraded to the lysosomes, chaperone and LAMP2A dependent. No ubiquitination involved.
What is microautophagy?
Another pathway involved in lysosomes. Ubiquitin independent.
What is macroautophagy?
Involves sequestration and transport of protein aggregates and organelles, within the membrane limited structures to the lysosomes for degradation. Can be ubiquitin dependent. Focus of the lecture.
What is the physiological significance of autophagy?
When nutrient status is low, non essential components can be recycled to provide energy and amino acids for synthesis of new essential proteins. Also involved in disease prevention. Carries out the removal of misfolded protein aggregates (pathogenic), damaged organelles, invading intracellular bacteria (e.g., salmonella that hides in cytoplasm).
What are the stages of macro autophagy?
Nucleation: Membrane isolation and phagophore formation.
Phagophore expansion.
Cargo recognition. (e..g, protein aggregates/intracellular bacteria/nucleus/mitochondria and many more substrates.).
Vesicle completion.
Fusion with lysosome.
Degradation and recycling.
Why are ribosomes degraded by autophagy?
To regulate protein synthesis. Idea that all pathways are communicating with eachother. Autophagy can also degrade the 26S proteosome.
How does ubiquitin bring selectivity in autophagy?
Developing autophagosome (phagophore). Protein aggregates or specific proteins on the surface of organelles/bacteria are ubiquitinated with K63-linked chains. Adaptor proteins (autophagy receptors) recognise the ub-modified cargoes. Cargo is incorporated into autophagosome by autophagy receptor. Can additionally bind to LC3II a membrane embedded protein with different domains to ubiquitin binding domains. Part of the ATG8 family.
What is the ATG8 protein family?
All are ubiquitin like in tertiary structure. Roles in autophagy.
What is the mechanism behind LC3 function?
Exists in two forms. LC3-I cytosolic form and LC3-II membrane bound form. LC3-I is converted into LC3-II through lipidation with phosphatidylethanolamine (PE). LC3-II is incorporated into the autophagosome membrane facilitating its expansion and maturation. LC3-II interacts with autophagy receptors which help recognize and sequester cargo for degradation. Recruits factors that mediate fusion between autophagosomes and lysosomes.
What do autophagy receptors do and name some examples.
Simultaneously bind to ubiquitin and LC3-II. Examples are p62, NDP52, OPTN, NBR1. The target binds to ubiquitin then LC3-II in the membrane for autophagy.
What are examples of increasing avidity with ubiquitin and autophagy receptors?
The receptors have single ubiquitin binding domains so cannot have polyubiquitination to increase avidity. Instead they oligomerise non-covalently. P62 and NBR1 have a PB1 domain which is an oligomerisation domain. NDP52 increases avidity through a coiled coil domain where the receptors wrap around eachother.
What is the structure of LIR motifs in comparison to the ubiquitin binding domains on autophagy receptors?
They are simple.
What is an additional avidity affect with LC3?
Restricted localisation of LC3 to the membrane.
How does Ser65-phosphorylation of ubiquitin amplify the ubiquitin signal?
Mitochondria are damaged and need to be degraded by autophagy. PINK1 (kinase) is expressed on the cell surface of mitochondria and gets activated. It is in a complex with Tom20. PINK1 has two substrates one is ubiquitin and at position 65 in the 76 residue there is a serine and ubiquitin is phosphorylated on serine 65 (phospho ubiquitination). PINK1 also phosphorylates an E3 ligase parkin. One of the domains is a UBL domain (ubiquitin like). In this at position 65 there is also a serine. Phosphorylation of this serine causes a confirmational change in parkin. Lets the E2 enzyme in and activates E3 ligase activity of Parkin. Builds polyubiquitin chains of the phosphorylated ubiquitin (lysine63 linked).
How are selectivity and avidity shown in the Ser65 phosphorylation?
Phospho ubiquitin chains are what are recognised with high affinity by autophagy receptors. Selectivity of these autophagy receptors because of the phosphorylation modification. Also acts as an inhibitor of DUBs.
How does further amplification by TBK1 occur?
Autophagy receptors oligomerise. TBK1 is a kinase and interacts with them and oligomerises to become activated. This starts phosphorylating autophagy receptors in two positions. UBD and LC3 interacting regions. Promotes affinity of UBD for phospho ubiquitin. Phosphorylation of LC3 increases affinity of LIR to LC3 protein.
