Degradation and Lysosome Flashcards
why do proteins need to be degraded?
- incomplete or missense proteins
- damaged proteins
- unwanted proteins
what are incomplete/missense proteins?
- cellular errors
- proteins with disruptive mutations
- premature termination
- proteolytic cleavage
what is post synthetic damage?
- misfolded proteins
- protein ageing
- denaturation
what are unwanted proteins?
- inactive or ‘used’ proteins
- free subunits or multimeric complexes
- other proteins made in excess
what are the two sites for degradation?
- Lysosome
- Proteasome
what is degraded in the proteasome?
soluble proteins (in the cytoplasm)
what is degraded in the lysosome?
soluble proteins/organelles (autophagy)/pathogens/membrane
what is the lysosome?
- membrane bound organelle
- contains 63 acidic hydrolases
- capable of degrade DNA, RNA, protein, carbohydrate, lipid
what is common to the proteasome and lysosome?
ubiquitin - which targets protein for degradation
- a protein will become ubiquinated and is then sent to the proteasome or lysosome
what is autophagy?
- when you eat yourself
- some material that is taken up for autophagy is ubiquinated
- autophagosome circularises the material
- devilvered to the lysosome
what is ubiquitin?
- 76 amino acid polypeptide that labels proteins
- attached by C terminus to tag the protein onto lysine residues
- very common in cells
how is ubiquitin added?
ubiquitin conjugating enzymes
what is E1?
ubiquitin activating enzyme: uses ATP to activate the ubiquitin
- passed from E1 to E2
what is E2?
- ubiquitin conjugating enzymes: passes the ubiquitin via E3 (ubiquitin ligase) to the substrate
what is E3?
- has a ring domain = ubiquitin is passed directly from E2 to substrate
- has a hect domain = ubiquitin is passed from E2 to the E3 to the substrate
what does ubiquitin attach to?
lysine residues
- ubquitin itself has lysine residues so ubiquitin can be added to ubiquitin
what is mono ubiquitin?
one ubiquitin
- one can be added to each lysine
- so can get multiple mono ubiquination
what is poly ubiquitin?
- ubiquitin in a chain
- ubiquitin itself can be ubiquinated
what does ubiquination determine?
- produces different structures which determines where and how its degraded
- cell recognises the differences
is ubiquitin reversible?
yes
what are DUBs?
- de-ubiquitnases
- ubiquitin is removed and recycled
- not generally degraded
- can save a protein from degradation with a DUB
what happens when you degraded the protein?
cell wants to use the Ub again, so its removed just before degradation
-requires DUB
what other signal can Ub provide?
- can have a non-degradation signal
- protein editing
- remove the signal
- requires DUB
where are ubiquitin synthesized and processed?
- synthesized on cystolic ribosomes
- rapidly processed into mature ubiquitin by DUBs
what is ubiquitin protein targeting?
- different Ub conformations will give a molecular tag
- enables Ub binding proteins to target the protein for degradation
how are proteins targeted to the proteasome?
- RPN10 and RPNB
- Shuttling receptors
what are RPN10 and RPNB?
- intrinsic receptors
- these are proteasomal proteins
- directly bind Ub cargo
- have Ub bidning domains
what are shuttling receptors?
- domains to the proteasome
- they have domains that bind Ub
- bring proteins to the proteasome
what are the majority of substrates for the proteasome?
- polyubiquinate (lys48)
- traditionally 4xlys48 are the minimal targetting motif
- LysII, Lys63 and mono ubiquitin epitopes can also be potential substrate
what are the target proteins in degradation via the proteasome?
- fed into the cap proteins
- denatured
- feed through proteolytic core
- short peptides then released
what are unfolding proteins?
- AAA_ATPase ‘cap’ proteins unfold proteins
- allows access to the proteolytic core
- hydrolysing ATP to unfold
what is an example of targetting proteins to the proteasome?
- tumour suppressor protein p53
- gene transcription of MDM2 and MDMx
- MDM2 –> E3 Ub ligase
- p53 gets degraded in the proteasome
- if you have less p53 you have less MDM2 which gives less degradation so p53 increases
- MDMX works in a similar loop
- auto inhibitory loop
- cell can regulate the levels of p53
- polyubuitination can cause targeting to the proteasome
what is the function of the lysosome?
degradation, autophagy, phagocytosis, disease
what is EGF receptor degradation?
- receptor mediated andocytosis and receptor binds ligand
- conformational change and phosphorylation
- receptor is ubiquinated
- in the early endosome you can still get signalling from this active receptor bound to ligand
- need to internalise the receptor so it can be degraded
what do you need to totally degrade a receptor?
-need to have all parts of the receptor available to the lysosomal hydrolases
what happens if the receptor isnt internalised when the endosome and lysosome fuse?
only degrades part of the protein
what is a MVB?
- a multivesicular body (a late endosome)
- invagination of the endosome surface
- end up with a vesicle inside the endosome itself
what happens when the late endosome and the lysosome fuse?
they can degrade absolutely everything and have stopped the signalling
how do you get internalisation of the limiting membrane?
- endosomal sorting complexes required for transport (ESCRT)
- proteins recognise membrane bound ubiquinated cargo and aids in their sorting into MVBs
what is the role of ESCRTs?
- Ub causes the binding of the different ESCRTs
- activation of the ESCRT complex proteins binds to Ub cargo causing membrane invagination and the cargo to be internalised
what does autophagy mean?
- process of self-cannibalisation
- cells capture their own cytoplasm and organelles and deliver them to the lysosome for nutrient release
- selective autophage often caputres ubiquinated cargo
what is autophagy a response to?
nutrient deprivation
- start eating ourselves for energy
what is the process of autophagy?
- proteins and carbohydrates in the cytoplas,
- double membrane surrounds the cytoplasm
- forms the autophagosome
- nucleation and extension
- closure
- fusion with lysosomes
- digestion
what is macro autophagy?
PAS - phagophore - autophagosome - autolysosome
mitophagy?
eating mitochondria
what is aggrephagy?
protein aggregate
what is pexophagy?
peroxisome
what does autophagy regulate?
energy homeostasis
what are the initiators of autophagy?
stagnantion, ammonia stress, damage, developmental cues
- material is degraded to the building blocks to regulate energy
what are the consequences of impaired autophagy?
- lack of nutrients
- aggregate formation
- immune activation
- ROS accumulation
- chronic infection
what happens if you dont have mitophagy?
- can remove damaged mitochondria
- get an increase in reactive oxygen species
- creates oxidative damage
what can not being able to remove aggregates lead to?
neurodegenerative disease
where can you find apoptosis?
- cytotoxic T cells (cause killing)
- during development
- removal of damaged cells
- tissue homeostasis
- elimination of pre-maligmant cells eg p53 pathway
- skin cells (always renewing)
- outer cells of eyes (renewal)
what is apoptosis?
regulated and rpogrammed cell death
- a controlled non-inflammatory event
what is necrosis?
- cell death following injury and results in inflammation
- contents of the cell bursts out
- body recognises that the cell contents shouldnt be out of the cell
what are the different processes of apoptosis?
- nuclear fragmentation
- cell fragments
- apoptotic body
- phagocytosis of the apoptotic bodies
what does apoptosis depended on?
intracellular proteolytic cascade that is mediated by caspases
what are the 3 types of caspases?
- intiators
- executioners
- inflammatory
what are the properties of initiators capsase?
- caspase 8,9
- have a large and a small subunit (protease domain)
- exists as an inactive domain
- when they bind/get a signal it causes the inactive monomers to dimerise
- have proteolytic activity (cleave each other)
- creates a conformational change and the protein folds up
- becomes an active caspase
- they go on to cleave the executioner caspases
what are executioner caspases?
- also have proteolytic activity
- cleave substrates inside the cell eg DNA and the cytoskeleton
- induces apoptosis
what is the structure of caspases?
- adaptor-binding domain
- 2 cleavage sites
- protease subunits (large and small)
what are caspases 8 and 10?
extrinsic pathway (have a Ded domain)
what are caspses 9 and 2?
intrinsic pathway (have a Card domain)
what do 8, 10, 9 and 2 caspases cleave?
the executioner caspases 3,6 and 7
what are caspases activated by?
- dimerization (initiators)
- proteolysis (effectors)
what happens when executioners are activated?
- apoptosis is virtually guaranteed
- only way to stop it is to digest caspases
- happens at the end of apoptosis
what is CAD?
an endonuclease
how is DNA fragmented?
- inactive CAD has an inhibitor (iCAD) bound to it (in normal cells)
- an executioner caspase cleaves the inhibitor releasing the CAD
- endonuclease can fragment the DNA
- CAD cleaves DNA between nucleosomes
How do caspases cause amplification?
- caspase activation leads to a caspase cascade which amplifies to apoptotic signal
- caspases cleave nuclear and cytoskeletal proteins
- caspases cleave and activate gelsolin including actin severing
what is the result of amplification?
nuclear fragmentation, disruption of the cytoskeleton, membrane blebbing and cell fragmentation
what is procaspase?
inactive form
what are the two pathways that lead to degradation?
intrinsic and extrinsic pathway
what is the extrinsic pathway?
- the cell recieves a signal from the extracellular environment to initiate apoptosis
- ligand binds to a receptor
- e.g. death ligands and receptors
what are the death receptors in the extrinsic pathway?
- initiate apoptosis following ligand binding
- fas
- TNF-RI
what are the ligands of the extrinsic pathway?
- FasL
- TNF alpha
- TRAIL
what do the death receptors contain?
a death domain
- which interact with adaptor proteins through mutual death domains
how is Fas an example of the extrinsic pathway?
- FasL binds to Fas
- Fas interacts with adaptor proteins via the FADD
- FAD complexes with pro-caspase 8 (a DED caspase)
- domains interact with each other
- forms DISC
- turns into active caspase 8 as more caspases are recruited
- leads to apoptosis
what is FADD?
Fas-assocaited death domain
what is DED?
death effector domain
what is DISC?
death inducing signal complex
how is Fas involved in influenza defence?
- infected cell presents a virus peptide on its cell surface with the histocompatability complex
- CTL induces apoptosis by binding FasL to Fas on target cell
how does HIV affect Fas?
- results in the loss of CD4+ T cells
- HIV gene causes overexpression of FasL
- allows it to interact with Fas on CD4 T cells
- induces non-infected cells to commit suicide
- failure in response of immune response
what is the intrinsic pathway?
- normally involves the release of cytochrome c from mitochondria
- induced by stress, damage or developmental cues
- involves Bcl-2 proteins
what stimulates the intrinsic pathway?
- cytochrome c is released
- binds Apaf-1
- Apaf-1 has an apoptotic domain the CARD domain
- causes the Apaf1 to assemble the apoptosome which recruits pro-caspase9 via its CCARD domain binding to the CAR domain of Apaf1
- binding of CARD domains allows the recruitment of procaspase 9 which also has a CARD domain
- causes dimerization, activation and apoptosis
what is the role of Bcl2 proteins?
regulation of the intrinsic pathway
what is the Bcl2 family?
- there are pro-apoptotic (BH3 and sometimes BH1 and BH2)
- there are anti-apoptotic members (BH4)
- there are activators, suppressors and depressors
- they have BH = Bcl2 homology domain
- balance between these proteins that determines apoptosis
what is the role of the BH3-only proteins?
thought to promote apoptosis by inhibiting anti-apoptosis Bcl2 family proteins
what is the role of inactivity in the intrinsic pathway?
- have active anti-apoptotic protein that inhibits proteins such as Bax and Bak
- found in the membrane
- means the cytochrome c is contained in the mitochondrial membrane
what happens when these proteins are activated?
- activated BH3 only proteins
- Bcl2 proteins form a pore in the membrane
- cytochrome c is released and can bind to Apaf1
what can inhibit apoptosis?
- extracellular survival factors
- growth/topic factor
- trophic factors
what are grow/trophic factors?
- they are transmembrane binding
- activation of PI-3 and AKT (PkB)
- this phosphorylates a Bcl2 protein called Bad
- causes it to bind a molecular chaperone
- therefore no longer binding to Bcl2 or BcIXL
- no longer have the open pore
what are trophic factors?
factor inducing differentiation/survival
how is there cross-over between the two pathways?
extrinsic pathway can activate the intrinsic pathway
how can the extrinsic pathway activate the intrinsic pathway?
• FasL Fas FAD caspase 8 activation executioner death
- This active caspase 8 can cleave a Bcl2 protein (Bid) and it becomes tBid
o This inhibits Bcl2 on the mitochondrial membrane to cause the release of cytochrome c
o Activates caspase 9 via apoptosome formation
