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Intercal - Advanced molecules, membranes and cells > Cell-substrate interactions 2 > Flashcards

Cell-substrate interactions 2 Flashcards

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Organic Chemistry 101 flashcards
1
Q

What makes the ECM

A

Proteases - endopeptidases (cleaves proteins)

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2
Q

What is the main protease type in the ECM

A

Matrix metalloproteinases - 24 of them

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3
Q

Where are matrix metalloproteinases found

A

Outside and transmembrane

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4
Q

What ion do matrix metalloproteinases need to

A

Zinc

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5
Q

What do metalloproteinases break down

A

Anything - not very specific

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6
Q

Are MMPs secreted in their active form and why

A

No not secreted in an active form - to prevent autodigestion

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7
Q

How are MMPs activated

A

Pro-peptide cleavage is required for MMP activation

Pro-peptide covers active side.

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8
Q

What inhibits metalloproteases

A

TIMP - tissue inhibitors of metalloproteinases

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9
Q

What resulted from a TIMP2 knockout

A

Expected more MMP2 activation but got inactive MMP2 instead.

Why - TIMP is an inhibitor and also needed for activation. MMP2 is activated by MMP14 (via cleavage), TIMP2 links MMP2 and MMP14 on the cell surface.

However too much of TIMP2 is inhibitory by binding to MMP14 which prevents cleavage of MMP2

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10
Q

What activates MMP14

A

Pro-protein convertases (furin)
pro-MMP2 –> MMP2

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11
Q

What inhibits furin

A

Alpha-1-antitrypsin

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12
Q

How does cancer use the ECM

A

Degrades it which helps it metastasise

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13
Q

Where does cancer usually grow

A

Behind basal lamina - it degrades this to metastasise –> degrades the endothelial basal lamina and spread via the blood

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14
Q

What is zymography

A

Method to measure MMP activity by denaturing them.
MMPs are active even when completely denatured - this is because it changes MMP shape and exposes the active site.

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15
Q

How does zymography work

A

Take the cell medium –> concentrate it –> use SDS PAGE –> get staining

The gel may contain MMP substrate so the assay isnt very specific as MMPs arent very specific but can tell MMP type via molecular weight

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16
Q

Give some pros of zymography

A

Easy
Cheap
Shows all the forms
Quantitative

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17
Q

Give some cons of zymography

A

Works best for secreted proteins
Requires millions of cells
Not cell-specific
Time consuming

18
Q

How does fluorescent based work

A

Put a thin layer of cells on fluorescent matrix with a cover plate - use microscope to look down - can see size, distribution and changes overtime

19
Q

Give some pros of fluorescent based

A

Cell specific
Quantitative
Provides localisation
Live

20
Q

Give some cons of fluorescent based

A

Requires microscope skill
Doesn’t identified proteases/MMPs - just identifies activity
Fluorescent matrix is expensive

21
Q

How does quenched based work

A

Flourescent dye is bound to peptide which MMPs cleave –> when cleaved the dye is quenched and becomes fluorescent –> can quantify how active the MMP is

22
Q

Give some pros of quenched based

A

Cheap
Easy
Fast
Quantitative

23
Q

Give some cons of quenched based

A

Require plate leader
Dont identify specific proteases
Not cell specific
Limited substrate available

24
Q

What is the transwell assay

A

A cell invasion type assay - simulate the process of mets -

Structure -
Cells and matrix
FILTER
Medium containing chemo-attractant

If cells can degrade matrix they will get through into chemo attractant - gives indication of ability and timeframe in vivo

25
Q

Pros of transwell assay

A

Easy to preform
Easy to analyse
Cheap
Quick

26
Q

Cons of transwell assay

A

Low reproducibility - too busy with too many variables to reproduce
Require chemo-attractant
Not cell specific
Lack of control of process

27
Q

What is 3D migration and how does it work

A

Method of looking at cell invasion - looking at cells while they degrade matrix.
Can add MMPs etc to look at result

28
Q

Give some pros of 3D migration

A

Very informative
Similar to vivo

29
Q

Give some cons of 3D migration

A

Complex to preform and analyse
Require instrumentations and skills
Expensive
Limitations on the possible matrix

30
Q

What other role do MMP have

WIDER READING - Sternlicht 2000

A

Cleave cell surface molecules to regulate cell behaviour - wound repair, inflammation and cancer

31
Q

What role do MMPs have in cancer

WIDER READING - Pozzi et al 2000

A

May initially increase the initial development of the cancer but overall decrease the severity.

32
Q

What role do MMPs have in arthritis

WIDER READING - (Mudgett et al. 1998).

A

Worsen it - stromelysin (MMP) deficient mice.

33
Q

How many MMPs are there

WIDER READING - Lohi et al 2001

A

22 in humans

34
Q

Give an example of MMP regulation regarding collagen

WIDER READING - Vogel et al 1997

A

Type I collagen acts as a ligand for discoidin domain-containing receptor-like tyrosine kinases that induce MMP1 expression when they are activated by intact collagen and become inactive when they bind MMP1-cleaved collagen

35
Q

How are MMPs released

WIDER READING - Raza et al 2000

A

Mostly automatically secreted once translated but In macrophages, plasmin and thrombin induce the secretion of MMP12, but do not alter its rate of transcription

36
Q

How is E-cadherin important in cancer

WIDER READING - Vleminckx 1991

A

E-cadherin is a calcium dependent transmembrane CAM and methylation of this occurs which silences it in cancer - this results in loss of cell adhesion allow mets

37
Q

What is the role of plakoglobin in cancer cells

WIDER READING - Aceto N et al 2014

A

CAM junction protein that when knocked-down in cancer resulted in less metastatic spread due to stopping cancer cell cluster formation

38
Q

How was zmyography used to determine laminin and MMP interactions in cancer cells

WIDER READING - Koshikawa 2000

A

Showed taht MT1-MMP is the trigger for laminin 5 migration as was shown to both be distributed over same area in the colon and breast cancer tissue specimens

39
Q

What is important to check for transwell assays

WIDER READING -F Trepat et al 2012

A

essential to check previously if cells are capable to invade the membrane and the Matrigel coating, as there exist cell types which can migrate horizontally very fast but they cannot invade a pore membrane

40
Q

Why cant prolonged transassays be done

Wider reading

A

Test agent will equalise between membrane.

Intercal - Advanced molecules, membranes and cells (31 decks)

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