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Cell Bio > Week 3 > Flashcards

Week 3 Flashcards

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Biology 101 flashcards
1
Q

What is ER stress

A

Occurs whenever the protein-folding capacity of the ER is overwhelmed

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2
Q

What is the rough ER associaated with

A

Biosynthesis of secretory and membrane proteins protien glycosylation and maturation and folding

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3
Q

What does the smooth ER associated with

A

lipid biosynthesis

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4
Q

What are the two organelles calcium is stored

A

ER and mitochondria

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5
Q

What is cehmical stimuli of ER stress

A

Biochemical inhibitors which can influence N-linked protien glycosylation, calcium homeostasis, vesicular transport

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6
Q

What are pathological conditions

A

Viral infenctions
Human disease

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7
Q

What does the drug Tunicamycin do?

A

Inhibitor of N-linked protien glycosylation in the ER

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8
Q

What is the dolichol phosphate

A

A long-chain polyisoprenoid lipid that is embedded into the ER membrane

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9
Q

What happens when the oligosaccharide precursor is synthesized

A

Flips to the ER lumen and becomes available for N-glycosylation fo relevant proteins

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10
Q

What is N-glycosylation

A

Covalent beta linkage of the precursor to the amide nitroge of the aspargine residue

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11
Q

What enzyme catalyzes core oligosaccharide

A

N-acetyleglucosaime

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12
Q

What does Tunicamycin act as

A

Tight binding competitive inhibitor close in comparison to UDP prevent the transfer of N-acetylglucosamine 1 phosphate to dolichoil monophosphate

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13
Q

What else can Tunicamycin do

A

Induce apoptosis

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14
Q

What type of inhibitor is Thapsigargin

A

Non-competitive of sacro-endoplasmic rectiulum Ca Atpase.

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15
Q

What are SERCAs

A

ATP driven ionic pumps which move Ca ions agianst gradient from the cytosol to the endoplasmic rectium remain high CA in the ER lumen

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16
Q

Where can Thapsigargin bind

A

Enzyme and enzyme substrate complex reduicng velocity of Ca

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17
Q

What is the Ca ions in the cytoplasm

A

100 nM

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18
Q

What is the cell concentration in ER

A

1 micro meters

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19
Q

What does calcium ions act in

A

Signal transduction and the increase in the cytoplasmic calcium activates a variety of signaling systems in cells

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20
Q

What is the homeostatic gradient of Ca is respobsible for

A

Essential for proper functioning fo the ER which important for proper folding of proeteins occurs only at a high local concentration of a Ca

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21
Q

What happens when cells are treated with Thapsigargin

A

Inhibit SERCA as a result of Ca ions will diffuse from the ER lumen into the cytoplasm

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22
Q

What is Brefeldin A

A

A macrocyclic lactone that inhibits vesicular transport between ER and Golgi

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23
Q

What is BFA

A

Targets and blocks the recruitment of a protein factor ARF1

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24
Q

What is ARF1

A

The small GTPase ADP-ribosylation factor 1which you need for COP1-coated vesicles

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25
Q

What is COP1 vesicles

A

They mediate retograde transport of KDEL with soluble proteins which are needed for ER to carrry out it’s function

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26
Q

What is KDEL

A

It is a sorting signal sequence at the C-terminal end of proteins which have to be located in the ER

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27
Q

What happens if COP1 transport is inhibited

A

The ER luminal proteins will not be returned to the ER and their recycling will be inhibited which increases the amount of unfolded proteins inducing the ER stress

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28
Q

What are some diseases releated to ER stress

A

neurodegeneration, stroke , bipolar disorder, cardiac, cancer, diabetes, muscle degeneration

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29
Q

What is the unfolded protein response

A

When an accumulation of unfolded proteins in the ER activates a series of homeostatic responses

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30
Q

What do cells do under mild stress conditions

A

Cell survival: refolding or elimination of misfolded proteins activate UPR mechanisms

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31
Q

What do cells do under severe stress

A

They trigger apoptosis

32
Q

What is BiP

A

and Hsp70 molecular chaperone also known as GRP78 a sensor for the ER stress pathway

33
Q

What does BiP do in unstressed cells

A

Mediates proper folding of proteins and keeps signalling molecules inactive

34
Q

What happens in with BiP in stressed cells

A

BiP preferentially binds to the unfolded proteins over the luminal domain of effectors the signalling molecules can signal to start the UPR cascade

35
Q

What are the three major effector branchers of the UPR

A

Insoitol- requring enzyme 1 (IRE1)
Protein kinase RNA-like ER kinase (PERK)
Activating transcrption factor 6 (ATF6)

36
Q

What do the three major effector branches of the UPR

A

Block general protein translation
Increase chaperone expression
Enhance ER-associated protein degradation

37
Q

What are the three domains of IRE1

A

Transmembrane domain
ER luminal domain
Cytosolic domain

38
Q

What is located in the ER luminal domain of IRE1

A

Binding BiP and an oligomerization site

39
Q

What is apart of the cytosolic domain

A

A protein serine/threonine kinase domain (phosphorylated different types of protiens)
endoribonuclease (RNAse) domain (cuts RNA)

40
Q

What is IRE1 considered

A

A bifunctional enzyme

41
Q

What are IRE1 substrates

A

IRE1 itself (trans-autophosphorylation)
mRNA molecule which encodes a transcription factor called XBP1

42
Q

What is the homolog of XBP1 in yeast

A

HAC1

43
Q

What is the process of singlaling of IRE1 in mild ER stress

A

BiP doesn’t bind too IRE1 which allows it to trans-autophosphorylation on the cytsoloic tail once is phosphorylates it lead to allosteric activation of the adjacent RNAase which then goes to splice a 26 intron from the unspliced XBP1. XBP1 now moves the nucleus and activates the transcprtion genes in UPR

44
Q

What is the process of signalling by IRE1 in severe ER stress

A

IRE1 oligomerizes and activates each other. The pathways it then goes to activate are 1. signalling pathways leading to apoptosis 2. promote degradation of miRNA targeting pro-apoptotic genes 3. promote the cleavage of various ER-localized mRNA

45
Q

What is the difference between oligomerized IRE1 and IRE1 dimers

A

Oligomerized IRE1 induced cell apoptosis while dimers induce cell adaptation

46
Q

How much longer is unspliced XBP1

A

26 nucleotides longer

47
Q

What test is used to detect XBP1

A

Cells are treated with thapsigargin and XBP1s is expressed more in treated cells in PCR

48
Q

What atare the three domains of PERK

A
  1. Transemembrane domain
  2. ER luminal domain (BiP and oligomerization)
  3. Cytosolic domian, Kinase and RNase
49
Q

What are PERKS substrates

A

PERK itself and elF2 alpha

50
Q

What is the signaling by PERK in association with Bip in unstressed cells

A

PERK is in a repressed state through an association with BiP

51
Q

What is the signaling by PERK in stressed cells

A

BiP release and dimerization of PERK which leads to trans-autophosphorylation of the cytoplasmic domain PERK than goes to phosphorylate elF2 which inihbits the elF2 activity and hence slows down global protein translation and ATF4 is upregulated can either be pro surival or prod death pathways

52
Q

What are pro survival genes

A

XBP1 chaperones and antioxidant enzymes

53
Q

What are pro death gene

A

which encodes a transcrption factor that inihibit the expression of antiapoptic BCL-2 and upregulate BIM mitohcondridal dependent apoptotic pathway

54
Q

What makes the structure of ATF6 different thatn IRE1 and PERK

A

The c terminal is in the ER luminal

55
Q

What are the three domains of ATF6

A
  1. Transmembrane domain
  2. ER luminal domain Bip binding domain and golgi localization sequences an golgi localization sequences
  3. Cytosolic domain which contains domains characterstic for transcrption factors bZIP and transcrption activation domain
56
Q

What is apart of the Golgi Localization Sequences

A

GLS1 and GLS2

57
Q

What can BiP bind too in ATF6

A

Only GLS1 and GLS2

58
Q

What is the signaling by ATF6 in unstressed cells

A

ATF6 is maintained in the ER through its association with BiP that binds to GLS1

59
Q

What is the signalling pathway of cells under ER stress

A

BiP is no longer bound too ATF6 so and GLS1 and GLS2 help move it to the Golgi in the Golgi ATF6 is the processed by Site-1 and Site-2 proteases releasing the cytosolic domain fragment ATF6f moves to the nucleus which can now upregulate UPR genes

60
Q

How can ATF6 translocation to the ER to the nucleus be detected

A

Fluroescence microscopy that have the GFP-ATF6 plasmid and you treat it with tunicamycin for different periods of time

61
Q

What does DAPI bind too

A

AT clusters

62
Q

What was observed in the fluroence microscopy

A

That untreated the ATF6 is located mostly in the cytoplasma and when treated is then found in the nucleus

63
Q

What is the steps to perform qPCR

A

Isolate total mRNA from control and treated cells, make cDNA and run qPCR using oligonucleotide primers specific to your gene of interest then you analyzed the expression of the genes

64
Q

What is BiP

A

GRP78

65
Q

What do the UPR response elements

A

ERSE
ERSE II
UPRE

66
Q

What do these response elements do

A

They bind the transcription factors of the ER stress but differes in specificity

67
Q

Does gene expression alway correlate with the expression of relevant proteins

A

NO

68
Q

What is western blotting

A

You move the electrophoresis transfer it to membrane apply a primary memebrany then the secondary antibody and then it can usally by chemiluminescence

69
Q

What was upregulated in the western blotting

A

CHOP sXBP1 phospho-elf2a and BiP

70
Q

What is imumunodetection

A

You are able to see where the actual protein is

71
Q

What is the process of direct antibody imumunodetection

A

Cells or tissues are fixed on glass slides and treated with specific antibodies the primary antibody is attached to an enzyme this is then detected by adding substrate. The antibody primary antibody is conjugated by fluorescence microscopy

72
Q

What is the down side of direct detection

A

Signal amplification is not possible the signal may be difficult to detect if the protein is in small quantities. There is also interference make it harder for the antibody to bind to it’s protien

73
Q

What is a benefit of the direct imumunodetection

A

You can use a variety of colors for furoscenets the only limit is how many colors the microscope can detect

74
Q

What is the indirect method of imumunodetection

A

It employs a secondary antibody with specificity against the unlabeled primary antibody to amplify the primary signal amplification is possible because of multiple secondary antibodies

75
Q

What is an advantage of the indirect immunostaining

A

As a higher specificity of immunodetection because the primary antibody is not chemically modified and can bind more strongley to the protien

76
Q
A

Cell Bio (8 decks)

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