TOPIC D: Protein Trafficking Flashcards
(100 cards)
1
Q
Where are many proteins made before performing their function?
A
- The cytosol
2
Q
Which 3 ways can proteins move across membranes?
A
- GATED TRANSPORT
- TRANSMEMBRANE TRASNPORT
- VESICULAR TRANSPORT
3
Q
What are 2 examples of transmembrane transport?
A
- cytosol–> mitochondira
- cytosol–> ER
4
Q
Do proteins have signals that tell them to move somewhere else?
A
- YES-SIGNAL SEQUENCES
- e.g. for import into ER it is hydrophobic stretch of aas (Leu-Val-Val) followed by BASIC aa
5
Q
What type of transport occurs INTO nucleus?
A
- GATED TRANSPORT of proteins
6
Q
What is the nucleus of the cell continuous with?
A
- The ER
7
Q
What does the nuclear lamina do?
A
- It is MESH and it STABILISES MEMBRANE
- encase DNA
- Proteins must be incorperated into lamina
8
Q
What is the nuclear pore complex comprised of ?
A
- cytosolic fibrils –> detect proteins that need to come in
- nuclear basket-> molecular sieve
9
Q
Which proteins can diffuse across the nuclear pore complex?
A
- proteins LESS than 50kDa in size e.g. GFP (Green Fluorescent Protein)
10
Q
How are proteins >50kDa in size transported through nuclear pores
A
ACTIVE TRANSPORT PROCESS –> efficient targeting
11
Q
What is the NLS? (Nuclear Localisation signal)
A
Basic Stretch of amino acids –> normal sequence
- Attaches to pyruvate dehydrogenase and goes into nucleus
12
Q
What is the structure that the proteins that are MORE THAN 50KDa (large) bind to in order to enter the nucleus?
A
- Via IMPORTINS (nuclear import receptors)
- They have a negative structure
13
Q
How can proteins moving into the nucleus be regulated?
A
- By making NLS (nuclear localisation signal)
14
Q
Which two ways can NLS be masked?
A
- Protein binding
2. Modification of /around the NLS
15
Q
What occurs in protein binding to unmask the NLS?
A
- 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
16
Q
What occurs in modification of/around the NLS to regulate it?
A
- Activation of phosphatase and inactivation of a kinase leads to DEPHOSPHORYLATION –> exposes NLS so importin can bind and transport into nucleus
17
Q
How are proteins incorporated into the nucleus (what conformation)?
A
- FOLDED
18
Q
Are importins soluble receptors?
A
- YES!
19
Q
How many proteins does mitochondrial DNA make?
A
- 13 proteins
20
Q
How do proteins move into the mitochondiral matrix?
A
- Proteins have N-terminal targeting signal (basic)
- It is an alpha helix (amphipathic -both hydro and hydryphillic parts)
21
Q
Once the proteins are in the mitochondria, what is chopped off?
A
- The targeting signal is clipped off
22
Q
What does TOM stand for?
A
- Translocase of outer membrane
23
Q
What does TIM stand for?
A
- Translocase of inner membrane
24
Q
What conformation must protein be in to cross TOM and TIM (double membranes of mito) into matrix of mito?
A
- UNFOLDED
25
Once protein is into mito matrix, what happens?
- Protein folded by CHAPERONES (via HSP70)
| - Targeting signal then cleaved off
26
What is required for import of proteins into mitochondira?
- A membrane potential
27
When does protein import into mitochondria occur?
- POST TRANSITIONALLY
28
What is the ER the major site of?
- Phospholipid biosynthesis
29
Are proteins transported into the ER co-translationally?
- YES!
30
Which particle is involved in the transportation of proteins into the ER?
- SRP (signal recognition particle) --> recognises a signal sequence on translating protein `
31
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
32
What is blocked to cause the buruli ulcer?
- THe ER translocon (because immune cells can't become active)
33
What is the secretory pathway?
- Constant flow of vesicle formation from ER--> Golgi--> outside of cell
34
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
35
What does oligosaccharyl transferase do?
- Transfers carbohydrate tree onto Asn (asparginine) residue --> amine group--> NH3--> N-linked
36
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
37
What is used to mark the state of protein folding?
- Oligosaccharides
38
What occurs if a protein is UNFOLDED?
- Precursor oligosaccharide binds--> 2 glucose molecules trimmed off--> protein partially folded
39
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
40
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
41
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!
42
What is a potential drug therapy for cystic fibrosis?
- ORKAMBI! (combination of 2 drugs)
43
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
44
What does vesicle trafficking maintain?
- Sidedness--> proteins stay inside and membrane proteins on outside
45
What is clathrin?
- Protein found in cytosol
| - binds to specific membranes and forms basket --> this forms vesicle
46
What structure assembles at the neck of the vesicle to cause budding?
- DYNAMIN (GTPase--> molecular scissors)
47
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)
48
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
49
Where is the KDEL receptor found?
- In the GOLGI and ER
50
What does the ER release proteins based on?
- pH gradient
51
What is the pH in the cis golgi network?
- LOW
52
What is the pH in the ER network?
- HIGH
53
What does the golgi do?
- Sorting compartment
- Helps mature proteins that have to be secreted
- Modifies glycosylation carbohydrates on proteins
54
What important process happens in the GOLGI?
- O-glycosylation (gives the O blood types) --> goes into sorting compartment for secretion
55
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)
56
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)
57
What does the constitutive pathway or regulated secretory pathway depend on?
- Cell and tissue type
58
What is the vesicle movement directed by?
-Motor proteins and microtubules (kinesins and dyenins bind vesicles and transport on MTS)
59
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
60
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
61
Which aspect of the lysosome allows enzymes to be active?
- H+ entering cell (Cl- transporter and ATP (V type) dependent proton pump--> pH=5)
62
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)
63
Which enzyme adds the phosphate to mannose?
- GlcNAc phosphotransferase adds the phosphate
64
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
65
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
66
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
67
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) `
68
What stimulates necrosis?
- PATHOGENS
69
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
70
What are three features of apoptosis?
- Cell shrinkage
- Nuclear blebbing
- Cell fragmentation into apoptotic bodies
71
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)
72
What are two ways that cells can apoptose?
1. Inside cell (suicide)--> DNA damage (UV), oxidative stress, cytosolic Ca2+ overload, unfolded protein accumulation
2. Outside cell (murder)--> Death signals from OTHER CELLS (Death ligands- Fas receptor )
73
What does the word CASPASE mean?
- Cystein ASPartic acid proteASES = CASPASES
74
Where are caspases present?
- In cytosol and nucleus
75
When do caspases cleave substrates after?
- Cleave after an aspartic acid
| `
76
In the first step of apoptosis, what are initiator caspases forced together by?
- Adaptor proteins (apoptotic signal)
| - Dimeristation
77
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
78
What are effector/executioner caspases activated by?
-Their own cleavage from initiator caspases
79
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.
80
What is iCAD?
- Inhibitor of Caspase Activated DNAse
| - DNAse inhibitor
81
Do caspases have to be MADE to induce apoptosis?
- NO! they are already there, they just have to be activated
82
Which two mechanisms are adaptor proteins made via?
1. Intrinsic pathway (mitochondrial) --> activarted caspase 9
2. Extrinsic Pathway (death receptor) pathway
(death receptor L--> death R--> TRADD--> FADD --> caspase 8 activated--> effector--> apoptosis)
83
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
84
what is the apoptosome made up of ?
- APAF-1 and cytochrome C
85
What important feature do you need for apaoptosome to form?
- Cytochrome C to come out of the mitochondria (cells committed to die)
86
What does cytochrome C play a major role in ?
- ETC (transfer of electrons)
87
Where does Cytochrome C normally sit?
- B/w outer and inner membrane of mitochondria
88
What is M.O.P?
- Mitochondrial Outermembrane Permeabalisation
| - Hole must be formed in OUTER membraen to let Cyt. C into cytosol of mito!
89
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
90
Which BCl2 family proteins are antiapoptotic?
- BCl2, BcIXL (Stops pore from letting cyt.C out)
91
which Bcl2 effector proteins are pro apoptotic?
- Bax, Bak (create pores in the outer mito. membrane to let Cyt.C out)
92
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
93
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!
94
What role do BH3 proteins have in cancer when mutated?
- Act as tumour suppressors
| e. g. Silencing of BIM common in B cell lymphomas
95
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
96
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
97
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
98
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
99
what does DISC stand for?
- Death Inducing Signalling Complex
100
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
