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BMS2021 > TOPIC D: Protein Trafficking > Flashcards

TOPIC D: Protein Trafficking Flashcards

1
Q

Where are many proteins made before performing their function?

A
  • The cytosol
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2
Q

Which 3 ways can proteins move across membranes?

A
  1. GATED TRANSPORT
  2. TRANSMEMBRANE TRASNPORT
  3. VESICULAR TRANSPORT
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3
Q

What are 2 examples of transmembrane transport?

A
  • cytosol–> mitochondira

- cytosol–> ER

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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

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5
Q

What type of transport occurs INTO nucleus?

A
  • GATED TRANSPORT of proteins
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6
Q

What is the nucleus of the cell continuous with?

A
  • The ER
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7
Q

What does the nuclear lamina do?

A
  • It is MESH and it STABILISES MEMBRANE
  • encase DNA
  • Proteins must be incorperated into lamina
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8
Q

What is the nuclear pore complex comprised of ?

A
  • cytosolic fibrils –> detect proteins that need to come in
  • nuclear basket-> molecular sieve
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9
Q

Which proteins can diffuse across the nuclear pore complex?

A
  • proteins LESS than 50kDa in size e.g. GFP (Green Fluorescent Protein)
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10
Q

How are proteins >50kDa in size transported through nuclear pores

A

ACTIVE TRANSPORT PROCESS –> efficient targeting

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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

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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

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13
Q

How can proteins moving into the nucleus be regulated?

A
  • By making NLS (nuclear localisation signal)
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14
Q

Which two ways can NLS be masked?

A
  1. Protein binding

2. Modification of /around the NLS

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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
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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
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17
Q

How are proteins incorporated into the nucleus (what conformation)?

A
  • FOLDED
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18
Q

Are importins soluble receptors?

A
  • YES!
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19
Q

How many proteins does mitochondrial DNA make?

A
  • 13 proteins
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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)

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21
Q

Once the proteins are in the mitochondria, what is chopped off?

A
  • The targeting signal is clipped off
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22
Q

What does TOM stand for?

A
  • Translocase of outer membrane
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23
Q

What does TIM stand for?

A
  • Translocase of inner membrane
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24
Q

What conformation must protein be in to cross TOM and TIM (double membranes of mito) into matrix of mito?

A
  • UNFOLDED
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25
Q

Once protein is into mito matrix, what happens?

A
  • Protein folded by CHAPERONES (via HSP70)

- Targeting signal then cleaved off

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26
Q

What is required for import of proteins into mitochondira?

A
  • A membrane potential
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27
Q

When does protein import into mitochondria occur?

A
  • POST TRANSITIONALLY
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28
Q

What is the ER the major site of?

A
  • Phospholipid biosynthesis
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29
Q

Are proteins transported into the ER co-translationally?

A
  • YES!
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30
Q

Which particle is involved in the transportation of proteins into the ER?

A
  • SRP (signal recognition particle) –> recognises a signal sequence on translating protein `
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31
Q

What is a stop-transfer sequence?

A
  • 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
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32
Q

What is blocked to cause the buruli ulcer?

A
  • THe ER translocon (because immune cells can’t become active)
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33
Q

What is the secretory pathway?

A
  • Constant flow of vesicle formation from ER–> Golgi–> outside of cell
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34
Q

What has to happen to a protein in the ER?

A
  • Must undergo folding and modifications
  • GLYCOSYLATION –> covalent attachment of carbohydrate to protein–> specific amino acid
  • In rough ER–> N-linked glycosylation
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35
Q

What does oligosaccharyl transferase do?

A
  • Transfers carbohydrate tree onto Asn (asparginine) residue –> amine group–> NH3–> N-linked
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36
Q

What is the role of glycosylation (4)?

A
  • Important in protein folding
  • Has a STABILISING role (e.g. in bloodstream)
  • Barrier function (protection from microbes)
  • Important for innate immune responses and antibodies
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37
Q

What is used to mark the state of protein folding?

A
  • Oligosaccharides
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38
Q

What occurs if a protein is UNFOLDED?

A
  • Precursor oligosaccharide binds–> 2 glucose molecules trimmed off–> protein partially folded
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39
Q

What happens to a protein if it is INCOMPLETELY FOLDED?

A
  • 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
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40
Q

Which gene is mutated in cystic fibrosis?

A
  • CFTR–> F508 deletion (Cl-) channel in membrane
  • Leads to mucous build up –> traps bacteria `
  • Misfolded protein stays in ER when not folded properly
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41
Q

If there was a mutation in the F508 channel (cystic fibrosis) BUT it got transported to the membrane would it be functional?

A
  • YES!

- It would have IMPAIRED function but still better than nothing!

42
Q

What is a potential drug therapy for cystic fibrosis?

A
  • ORKAMBI! (combination of 2 drugs)
43
Q

Which two drugs are part of ORKAMBI and what do they do?

A

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
Q

What does vesicle trafficking maintain?

A
  • Sidedness–> proteins stay inside and membrane proteins on outside
45
Q

What is clathrin?

A
  • Protein found in cytosol

- binds to specific membranes and forms basket –> this forms vesicle

46
Q

What structure assembles at the neck of the vesicle to cause budding?

A
  • DYNAMIN (GTPase–> molecular scissors)
47
Q

What pathway do our cells use to ensure no proteins escape into the secretory pathway?

A
  • retrieval pathway via KDEL (C terminal KDEL in ER proteins)
48
Q

What do the KDEL (4 aas) do and what happens when an ER protein accidentally exported ?

A
  • 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
Q

Where is the KDEL receptor found?

A
  • In the GOLGI and ER
50
Q

What does the ER release proteins based on?

A
  • pH gradient
51
Q

What is the pH in the cis golgi network?

A
  • LOW
52
Q

What is the pH in the ER network?

A
  • HIGH
53
Q

What does the golgi do?

A
  • Sorting compartment
  • Helps mature proteins that have to be secreted
  • Modifies glycosylation carbohydrates on proteins
54
Q

What important process happens in the GOLGI?

A
  • O-glycosylation (gives the O blood types) –> goes into sorting compartment for secretion
55
Q

Which 3 pathways can proteins undergo when exiting golgi?

A
  • LYSOSOME (signal mediated)
  • PLASMA MEMBRANE (signal mediated—> diversion to secretory vesicles)
  • SECRETORY VESICLE (constitutive secretory pathway)
56
Q

What is the difference between consitutive and regulatory secretory pathway?

A
  • 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
Q

What does the constitutive pathway or regulated secretory pathway depend on?

A
  • Cell and tissue type
58
Q

What is the vesicle movement directed by?

A

-Motor proteins and microtubules (kinesins and dyenins bind vesicles and transport on MTS)

59
Q

What is the mechanism of lysosomes?

A
  • Contain hydrolytic enzymes that are active under ACIDIC CONDITIONS maintained by a proton pump. e.g. Nucleases, proteases, glycosylases
60
Q

Are hydrolytic enzymes active in the ER and golgi?

A
  • NO! (ER doesn’t have low pH for them to work) Imported to ER, trafficked to golgi, sorted to lysosomes
61
Q

Which aspect of the lysosome allows enzymes to be active?

A
  • H+ entering cell (Cl- transporter and ATP (V type) dependent proton pump–> pH=5)
62
Q

How are the lysosomal proteins degraded?

A
  • Glycosylated in ER –> phosphate added to C6 in mannose binding sugar in GOLGI –> Mannose-6-P recognised in (directed to lysosome)
63
Q

Which enzyme adds the phosphate to mannose?

A
  • GlcNAc phosphotransferase adds the phosphate
64
Q

What happens when there is a defficiency in GlcNAc phosphotransferase?

A
  • 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
Q

What is an example of defficiency in GlcNAc phosphotransferase?

A
  • 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
Q

What is familial hypercholesterolemia? (don’t need to know for exam)

A
  • 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
Q

What is necrosis?

A
  • 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
Q

What stimulates necrosis?

A
  • PATHOGENS
69
Q

What is apoptosis?

A
  • 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
Q

What are three features of apoptosis?

A
  • Cell shrinkage
  • Nuclear blebbing
  • Cell fragmentation into apoptotic bodies
71
Q

In grown people, for every cell division what do you need?

A
  • A cell death via APOPTOSIS e.g. T cell selection (neurons that don’t connect)
72
Q

What are two ways that cells can apoptose?

A
  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
Q

What does the word CASPASE mean?

A
  • Cystein ASPartic acid proteASES = CASPASES
74
Q

Where are caspases present?

A
  • In cytosol and nucleus
75
Q

When do caspases cleave substrates after?

A
  • Cleave after an aspartic acid

`

76
Q

In the first step of apoptosis, what are initiator caspases forced together by?

A
  • Adaptor proteins (apoptotic signal)

- Dimeristation

77
Q

Once initiator caspases are activated, what happens?

A
  • 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
Q

What are effector/executioner caspases activated by?

A

-Their own cleavage from initiator caspases

79
Q

How do caspases control demolition of cell?

A
  • Condensation of nulcear DNA and fragmentation

inactive CAD bound to iCAD–> ICAD cleaved–> active CAD–> DNA fragmentation etc.

80
Q

What is iCAD?

A
  • Inhibitor of Caspase Activated DNAse

- DNAse inhibitor

81
Q

Do caspases have to be MADE to induce apoptosis?

A
  • NO! they are already there, they just have to be activated
82
Q

Which two mechanisms are adaptor proteins made via?

A
  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
Q

What are the details of how caspase 9 is activated?

A
  • 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
Q

what is the apoptosome made up of ?

A
  • APAF-1 and cytochrome C
85
Q

What important feature do you need for apaoptosome to form?

A
  • Cytochrome C to come out of the mitochondria (cells committed to die)
86
Q

What does cytochrome C play a major role in ?

A
  • ETC (transfer of electrons)
87
Q

Where does Cytochrome C normally sit?

A
  • B/w outer and inner membrane of mitochondria
88
Q

What is M.O.P?

A
  • Mitochondrial Outermembrane Permeabalisation

- Hole must be formed in OUTER membraen to let Cyt. C into cytosol of mito!

89
Q

What is BCL-2 and what is its mechanism of action?

A
  • 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
Q

Which BCl2 family proteins are antiapoptotic?

A
  • BCl2, BcIXL (Stops pore from letting cyt.C out)
91
Q

which Bcl2 effector proteins are pro apoptotic?

A
  • Bax, Bak (create pores in the outer mito. membrane to let Cyt.C out)
92
Q

What are the pro apoptotic BH2 only proteins?

A
  • Bad, Bim (blocks BCl-2), Bid, Noxa, Puma

- BIM competitively binds to BAX to block BCl2 from binding

93
Q

Where does BAX go after activation?

A
  • 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
Q

What role do BH3 proteins have in cancer when mutated?

A
  • Act as tumour suppressors

e. g. Silencing of BIM common in B cell lymphomas

95
Q

What is a way to try and treat cancers with a BH3 mutation?

A
  • 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
Q

What does BCI-XI regulate?

A
  • 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
Q

What does Venetoclax do?

A
  • Binds BCI-XI 3x WEAKER than BCl-2 (reducing symptoms)–> to MAINTAIN platelet levels in body and prevent thrombocytopenia WHILST treating cancer
98
Q

What are the details of extrinsic cell death?

A
  • 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
Q

what does DISC stand for?

A
  • Death Inducing Signalling Complex
100
Q

Why is the caspase 8 death (extracellular) less effective as cancer treatment than caspase 9?

A
  • 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

BMS2021 (7 decks)

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