TOPIC D: Protein Trafficking Flashcards
Where are many proteins made before performing their function?
- The cytosol
Which 3 ways can proteins move across membranes?
- GATED TRANSPORT
- TRANSMEMBRANE TRASNPORT
- VESICULAR TRANSPORT
What are 2 examples of transmembrane transport?
- cytosol–> mitochondira
- cytosol–> ER
Do proteins have signals that tell them to move somewhere else?
- YES-SIGNAL SEQUENCES
- e.g. for import into ER it is hydrophobic stretch of aas (Leu-Val-Val) followed by BASIC aa
What type of transport occurs INTO nucleus?
- GATED TRANSPORT of proteins
What is the nucleus of the cell continuous with?
- The ER
What does the nuclear lamina do?
- It is MESH and it STABILISES MEMBRANE
- encase DNA
- Proteins must be incorperated into lamina
What is the nuclear pore complex comprised of ?
- cytosolic fibrils –> detect proteins that need to come in
- nuclear basket-> molecular sieve
Which proteins can diffuse across the nuclear pore complex?
- proteins LESS than 50kDa in size e.g. GFP (Green Fluorescent Protein)
How are proteins >50kDa in size transported through nuclear pores
ACTIVE TRANSPORT PROCESS –> efficient targeting
What is the NLS? (Nuclear Localisation signal)
Basic Stretch of amino acids –> normal sequence
- Attaches to pyruvate dehydrogenase and goes into nucleus
What is the structure that the proteins that are MORE THAN 50KDa (large) bind to in order to enter the nucleus?
- Via IMPORTINS (nuclear import receptors)
- They have a negative structure
How can proteins moving into the nucleus be regulated?
- By making NLS (nuclear localisation signal)
Which two ways can NLS be masked?
- Protein binding
2. Modification of /around the NLS
What occurs in protein binding to unmask the NLS?
- TF-BP (Transcription factor binding protein) is degraded and exposes the NLS on the transcription factor (which is +ve) –> NLS can now be exported into nucleus via importin
What occurs in modification of/around the NLS to regulate it?
- Activation of phosphatase and inactivation of a kinase leads to DEPHOSPHORYLATION –> exposes NLS so importin can bind and transport into nucleus
How are proteins incorporated into the nucleus (what conformation)?
- FOLDED
Are importins soluble receptors?
- YES!
How many proteins does mitochondrial DNA make?
- 13 proteins
How do proteins move into the mitochondiral matrix?
- Proteins have N-terminal targeting signal (basic)
- It is an alpha helix (amphipathic -both hydro and hydryphillic parts)
Once the proteins are in the mitochondria, what is chopped off?
- The targeting signal is clipped off
What does TOM stand for?
- Translocase of outer membrane
What does TIM stand for?
- Translocase of inner membrane
What conformation must protein be in to cross TOM and TIM (double membranes of mito) into matrix of mito?
- UNFOLDED
Once protein is into mito matrix, what happens?
- Protein folded by CHAPERONES (via HSP70)
- Targeting signal then cleaved off
What is required for import of proteins into mitochondira?
- A membrane potential
When does protein import into mitochondria occur?
- POST TRANSITIONALLY
What is the ER the major site of?
- Phospholipid biosynthesis
Are proteins transported into the ER co-translationally?
- YES!
Which particle is involved in the transportation of proteins into the ER?
- SRP (signal recognition particle) –> recognises a signal sequence on translating protein `
What is a stop-transfer sequence?
- Transmembrane anchor (very hydrophobic)
- When protein being fed through TRANSLOCON it is HYDROPHILLIC but when it comes across HYSORPHOBIC part (STOP-transfer) it DOESN’T LIKE IT
- Translocon opens up and lets protein OUT into ER membrane
What is blocked to cause the buruli ulcer?
- THe ER translocon (because immune cells can’t become active)
What is the secretory pathway?
- Constant flow of vesicle formation from ER–> Golgi–> outside of cell
What has to happen to a protein in the ER?
- Must undergo folding and modifications
- GLYCOSYLATION –> covalent attachment of carbohydrate to protein–> specific amino acid
- In rough ER–> N-linked glycosylation
What does oligosaccharyl transferase do?
- Transfers carbohydrate tree onto Asn (asparginine) residue –> amine group–> NH3–> N-linked
What is the role of glycosylation (4)?
- Important in protein folding
- Has a STABILISING role (e.g. in bloodstream)
- Barrier function (protection from microbes)
- Important for innate immune responses and antibodies
What is used to mark the state of protein folding?
- Oligosaccharides
What occurs if a protein is UNFOLDED?
- Precursor oligosaccharide binds–> 2 glucose molecules trimmed off–> protein partially folded
What happens to a protein if it is INCOMPLETELY FOLDED?
- CALNEXIN (chaperone) binds to glucose on N-linked oligosaccharide–> holds onto protein and makes sure it starts to fold–> sent out in vesicles IF FOLDED
OR
ReAttaches glucose oligosaccharide–> trimmed –> folds properly–> exit out of ER
Which gene is mutated in cystic fibrosis?
- CFTR–> F508 deletion (Cl-) channel in membrane
- Leads to mucous build up –> traps bacteria `
- Misfolded protein stays in ER when not folded properly
If there was a mutation in the F508 channel (cystic fibrosis) BUT it got transported to the membrane would it be functional?
- YES!
- It would have IMPAIRED function but still better than nothing!
What is a potential drug therapy for cystic fibrosis?
- ORKAMBI! (combination of 2 drugs)
Which two drugs are part of ORKAMBI and what do they do?
LUMACAFTOR= CFTR CORECEPTOR
- assists in folding of F508 deletion CFTR and INCREASE amount of chaperone activity
IVACAFTOR= CFTR POTENTIATOR
- Increase in opening probability of CFTR channels
What does vesicle trafficking maintain?
- Sidedness–> proteins stay inside and membrane proteins on outside
What is clathrin?
- Protein found in cytosol
- binds to specific membranes and forms basket –> this forms vesicle
What structure assembles at the neck of the vesicle to cause budding?
- DYNAMIN (GTPase–> molecular scissors)
What pathway do our cells use to ensure no proteins escape into the secretory pathway?
- retrieval pathway via KDEL (C terminal KDEL in ER proteins)
What do the KDEL (4 aas) do and what happens when an ER protein accidentally exported ?
- Lets the cell know that it is an ER protein and if it escapes, how to get back into the ER
- When ER protein accidentally exported, it goes into vesicle–> binds to KDEL receptor in GOLGI–> repackages it –> sends it back to ER
Where is the KDEL receptor found?
- In the GOLGI and ER
What does the ER release proteins based on?
- pH gradient
What is the pH in the cis golgi network?
- LOW
What is the pH in the ER network?
- HIGH
What does the golgi do?
- Sorting compartment
- Helps mature proteins that have to be secreted
- Modifies glycosylation carbohydrates on proteins
What important process happens in the GOLGI?
- O-glycosylation (gives the O blood types) –> goes into sorting compartment for secretion
Which 3 pathways can proteins undergo when exiting golgi?
- LYSOSOME (signal mediated)
- PLASMA MEMBRANE (signal mediated—> diversion to secretory vesicles)
- SECRETORY VESICLE (constitutive secretory pathway)
What is the difference between consitutive and regulatory secretory pathway?
- REGULATED–> Ligand must bind to cause release (e.g. insulin sitting packed in vesicles)
- CONSTITUTIVE–> Make proteins and secrete without any regulation (just need to get sent out e.g. antibodies)
What does the constitutive pathway or regulated secretory pathway depend on?
- Cell and tissue type
What is the vesicle movement directed by?
-Motor proteins and microtubules (kinesins and dyenins bind vesicles and transport on MTS)
What is the mechanism of lysosomes?
- Contain hydrolytic enzymes that are active under ACIDIC CONDITIONS maintained by a proton pump. e.g. Nucleases, proteases, glycosylases
Are hydrolytic enzymes active in the ER and golgi?
- NO! (ER doesn’t have low pH for them to work) Imported to ER, trafficked to golgi, sorted to lysosomes
Which aspect of the lysosome allows enzymes to be active?
- H+ entering cell (Cl- transporter and ATP (V type) dependent proton pump–> pH=5)
How are the lysosomal proteins degraded?
- Glycosylated in ER –> phosphate added to C6 in mannose binding sugar in GOLGI –> Mannose-6-P recognised in (directed to lysosome)
Which enzyme adds the phosphate to mannose?
- GlcNAc phosphotransferase adds the phosphate
What happens when there is a defficiency in GlcNAc phosphotransferase?
- NO mannose-6-P formed (N-acetyl glucosamine P–> mannose in oligosaccharide DOES NOT HAPPEN)
- Lysosomal hydrolase enzymes secreted from cells rather than targeted to lysosomes
What is an example of defficiency in GlcNAc phosphotransferase?
- I-cell disease (i cell) –> lysosomal storage disorder
- non functional enzymes
- Lysosome degradation doesn’t work (build up of lysosomes in cell )
- Symptoms of short skeletal abnormalities, cardiomegaly, developmental delay
What is familial hypercholesterolemia? (don’t need to know for exam)
- HIGH blood cholesterol levels
- Mutation in genes –> LDL receptor not being produced
- LDL R can’t bind LDL
- LDL R binding but can’t internalise
- HIGH RISK of heart attack
What is necrosis?
- Cells that die via release of DAMPs
- Necrotic cells trigger inflammation by innate immune cells (e.g. If you cut finger, cells will die via necrosis) `
What stimulates necrosis?
- PATHOGENS
What is apoptosis?
- Death from within –> no spillage of cell contents like necrosis
- cell membrane stays intact and ‘blebs’ off into smaller bodies –> engulfed by phagocytes into lysosomes and recycled
What are three features of apoptosis?
- Cell shrinkage
- Nuclear blebbing
- Cell fragmentation into apoptotic bodies
In grown people, for every cell division what do you need?
- A cell death via APOPTOSIS e.g. T cell selection (neurons that don’t connect)
What are two ways that cells can apoptose?
- Inside cell (suicide)–> DNA damage (UV), oxidative stress, cytosolic Ca2+ overload, unfolded protein accumulation
- Outside cell (murder)–> Death signals from OTHER CELLS (Death ligands- Fas receptor )
What does the word CASPASE mean?
- Cystein ASPartic acid proteASES = CASPASES
Where are caspases present?
- In cytosol and nucleus
When do caspases cleave substrates after?
- Cleave after an aspartic acid
`
In the first step of apoptosis, what are initiator caspases forced together by?
- Adaptor proteins (apoptotic signal)
- Dimeristation
Once initiator caspases are activated, what happens?
- They bind onto EXECUTIONER CASPASES (effector caspases)
- Initiator caspases CLEAVE EXECUTIONER CASPASES TO ACTIVATE THEM –> Then multiple substrates are cleaved (degradation of DNA and cytskeleton)–> apoptosis
What are effector/executioner caspases activated by?
-Their own cleavage from initiator caspases
How do caspases control demolition of cell?
- Condensation of nulcear DNA and fragmentation
inactive CAD bound to iCAD–> ICAD cleaved–> active CAD–> DNA fragmentation etc.
What is iCAD?
- Inhibitor of Caspase Activated DNAse
- DNAse inhibitor
Do caspases have to be MADE to induce apoptosis?
- NO! they are already there, they just have to be activated
Which two mechanisms are adaptor proteins made via?
- Intrinsic pathway (mitochondrial) –> activarted caspase 9
- Extrinsic Pathway (death receptor) pathway
(death receptor L–> death R–> TRADD–> FADD –> caspase 8 activated–> effector–> apoptosis)
What are the details of how caspase 9 is activated?
- Inactive caspase 9 is forced to dimerise on APOPTOSOME (made up of APAF-1 and Cyt.C)
- Activation of APAF1 –> both APAF1 and Cyt.C bind to wheel of apoptosome –> forces Caspase 9 to dimerise after binding to CARD signal —> ACTIVATED WHEEL AND ACTIVATED CASPASE 9 –> cleacves effector caspases to make them active
what is the apoptosome made up of ?
- APAF-1 and cytochrome C
What important feature do you need for apaoptosome to form?
- Cytochrome C to come out of the mitochondria (cells committed to die)
What does cytochrome C play a major role in ?
- ETC (transfer of electrons)
Where does Cytochrome C normally sit?
- B/w outer and inner membrane of mitochondria
What is M.O.P?
- Mitochondrial Outermembrane Permeabalisation
- Hole must be formed in OUTER membraen to let Cyt. C into cytosol of mito!
What is BCL-2 and what is its mechanism of action?
- Protein that inhibits apoptosis (anti apoptotic) and is found on the outer mito membrane .
- Prevents apoptosis by preventing Bax and Bak from forming a pore in the membrane thus Cyt. C can’t go out and induce apooptosis
Which BCl2 family proteins are antiapoptotic?
- BCl2, BcIXL (Stops pore from letting cyt.C out)
which Bcl2 effector proteins are pro apoptotic?
- Bax, Bak (create pores in the outer mito. membrane to let Cyt.C out)
What are the pro apoptotic BH2 only proteins?
- Bad, Bim (blocks BCl-2), Bid, Noxa, Puma
- BIM competitively binds to BAX to block BCl2 from binding
Where does BAX go after activation?
- To MITO oouter membrane
- Drives oligomers–> makes pore in membrane –> cytochrome C OUT in cytosol
- binds to APAF-1
- Apoptosome–> caspase 9 activated –> drives Caspase 3–> cell death!
What role do BH3 proteins have in cancer when mutated?
- Act as tumour suppressors
e. g. Silencing of BIM common in B cell lymphomas
What is a way to try and treat cancers with a BH3 mutation?
- Try to reintroduce BH3 only mimetic
- binds to BCl-2 (antiapoptotic)
- NEEDS TO BE SOLUBLE TO BE USED AS A THERAPY –> e.g. Nantoclax
What does BCI-XI regulate?
- Platelet life span (blood clotting and wound healing)
- Life span dependent on BCI-XI regulating actions of BAK/BAX to prevent death occurring TOO EARLY
- WE NEED A BH3 MIMETIC TO BIND BCL-2 NOT BCIXI
What does Venetoclax do?
- Binds BCI-XI 3x WEAKER than BCl-2 (reducing symptoms)–> to MAINTAIN platelet levels in body and prevent thrombocytopenia WHILST treating cancer
What are the details of extrinsic cell death?
- Ligand (TNF or Fas) binds to receptor–> cytosolic domain recruits adaptor proteins –> FADD which binds and ACTIVATES initiator caspase 8 –> forms DICS (Death Inducing Signalling Complex) –> active caspase 8 activates execution caspases
what does DISC stand for?
- Death Inducing Signalling Complex
Why is the caspase 8 death (extracellular) less effective as cancer treatment than caspase 9?
- Because Caspase 9 activates tBID (BH3 only protein)
- tBID regulates BCL-2 family of proteins to activate pathway
- So targeting caspase 8 will cause down regulation of apoptosis from mitochondria pathway
