Cell-substrate interactions 2

Question 1

What makes the ECM

Answer 1

Proteases - endopeptidases (cleaves proteins)

Question 2

What is the main protease type in the ECM

Answer 2

Matrix metalloproteinases - 24 of them

Question 3

Where are matrix metalloproteinases found

Answer 3

Outside and transmembrane

Question 4

What ion do matrix metalloproteinases need to

Answer 4

Zinc

Question 5

What do metalloproteinases break down

Answer 5

Anything - not very specific

Question 6

Are MMPs secreted in their active form and why

Answer 6

No not secreted in an active form - to prevent autodigestion

Question 7

How are MMPs activated

Answer 7

Pro-peptide cleavage is required for MMP activation

Pro-peptide covers active side.

Question 8

What inhibits metalloproteases

Answer 8

TIMP - tissue inhibitors of metalloproteinases

Question 9

What resulted from a TIMP2 knockout

Answer 9

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

Question 10

What activates MMP14

Answer 10

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

Question 11

What inhibits furin

Answer 11

Alpha-1-antitrypsin

Question 12

How does cancer use the ECM

Answer 12

Degrades it which helps it metastasise

Question 13

Where does cancer usually grow

Answer 13

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

Question 14

What is zymography

Answer 14

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.

Question 15

How does zymography work

Answer 15

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

Question 16

Give some pros of zymography

Answer 16

Easy
Cheap
Shows all the forms
Quantitative

Question 17

Give some cons of zymography

Answer 17

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

Question 18

How does fluorescent based work

Answer 18

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

Question 19

Give some pros of fluorescent based

Answer 19

Cell specific
Quantitative
Provides localisation
Live

Question 20

Give some cons of fluorescent based

Answer 20

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

Question 21

How does quenched based work

Answer 21

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

Question 22

Give some pros of quenched based

Answer 22

Cheap
Easy
Fast
Quantitative

Question 23

Give some cons of quenched based

Answer 23

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

Question 24

What is the transwell assay

Answer 24

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

Question 25

Pros of transwell assay

Answer 25

Easy to preform Easy to analyse Cheap Quick

Question 26

Cons of transwell assay

Answer 26

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

Question 27

What is 3D migration and how does it work

Answer 27

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

Question 28

Give some pros of 3D migration

Answer 28

Very informative Similar to vivo

Question 29

Give some cons of 3D migration

Answer 29

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

Question 30

What other role do MMP have WIDER READING - Sternlicht 2000

Answer 30

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

Question 31

What role do MMPs have in cancer WIDER READING - Pozzi et al 2000

Answer 31

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

Question 32

What role do MMPs have in arthritis WIDER READING - (Mudgett et al. 1998).

Answer 32

Worsen it - stromelysin (MMP) deficient mice.

Question 33

How many MMPs are there WIDER READING - Lohi et al 2001

Answer 33

22 in humans

Question 34

Give an example of MMP regulation regarding collagen WIDER READING - Vogel et al 1997

Answer 34

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

Question 35

How are MMPs released WIDER READING - Raza et al 2000

Answer 35

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

Question 36

How is E-cadherin important in cancer WIDER READING - Vleminckx 1991

Answer 36

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

Question 37

What is the role of plakoglobin in cancer cells WIDER READING - Aceto N et al 2014

Answer 37

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

Question 38

How was zmyography used to determine laminin and MMP interactions in cancer cells WIDER READING - Koshikawa 2000

Answer 38

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

Question 39

What is important to check for transwell assays WIDER READING -F Trepat et al 2012

Answer 39

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

Question 40

Why cant prolonged transassays be done Wider reading

Answer 40

Test agent will equalise between membrane.