Chapter 7: Apoptosis (Secondary details)

Question 1

What is another word for apoptosis?

Answer 1

Programmed cell death (PCD)

Question 2

Macrophages are important in cleaning of cell debris. How do macrophages recognize a cell undergoing apoptosis?

Answer 2

They recognize molecular flags like phosphatidyl serine.

Question 3

Is apoptosis ATP dependent?

Answer 3

Yes, it is an active ATP dependent process.

Question 4

Just know that there are approximately 2,2 kg cells per day that go into apoptosis (this is part of homeostasis, where 25x10^6 mitoses per second occurs, there’s also the same amount of apoptosis per second)

Answer 4

Ok

Question 5

What is zeiosis?

Answer 5

Blebbing of the cell

Question 6

What is MGG-staining (don’t learn by heart)?

Answer 6

May Grunwald-Giemsa (MGG) Stain is used for staining of blood, bone marrow smears and clinical cytological specimens and is used for morphological inspection and differential counting of blood cells.

Question 7

What is Dapi staining (don’t learn by heart)?

Answer 7

Dapi is a fluorescent substance that binds to chromatin. When bound to chromatin it emits a cyan blue color.

Question 8

Besides Fas and TNF, there’s another ligand that can induce apoptosis via the extrinsic pathway. How is this ligand called and what is special about this ligand and its receptor?

Answer 8

TNF-related apoptosis-inducing ligand (TRAIL), this ligand has multiple receptors (TRAIL receptor 1, 2 …)

Question 9

What are decoy receptors (DC1-DC4) of the extrinsic pathway?

Answer 9

Receptors that for example have no intracellular domain and therefore cannot send an apoptotic/death signal downstreams.

Question 10

What is formed when BH3 proteins Bax oligomerizes on the outer mitochondrial membrane?

Answer 10

Pores, so that other proteins can be transported through these pores (it makes it more permeabel).

Question 11

What is typical about the extrinsic pathway? And what is typical about the intrinsic pathway?

Answer 11

Extrinsic pathway is characterized by the activation of caspases. The intrinsic pathway is characterized by the presence of the apoptosome that activates caspases.

Question 12

True/false: The Bcl-2 family is a large family of proteins with sequence homology.

Answer 12

True

Question 13

How can proteins of the Bcl-2 family form hetero- or homodimers?

Answer 13

Through shared domains (Bcl-2 homology domain BH1, BH2, BH3, BH4 or TM).

Question 14

How is determined whether Bcl-2 proteins have anti- or pro-apoptotic properties? What is an exception for this?

Answer 14

Through their shared domains (BH1/BH2/BH3/BH4/TM).

Most pro-apoptotic Bcl-2 proteins miss the BH4 domain, most anti-apoptotic Bcl-2 proteins have all the domains. An exception of this are pro-apoptotic Bcl-2 proteins that only contain BH3 (BH3-only proteins).

Question 15

How do the BH3-only proteins fullfill their role as pro-apoptotic Bcl-2 proteins?

Answer 15

They bind and inhibit anti-apoptotic Bcl-2 proteins.

Question 16

I don’t think (and hope) you need to learn this by heart. So just have an idea of the different Bcl-2 proteins and look at this table.

Answer 16

Ok

Question 17

Name 4 structures found in the mitochondria.

Answer 17

Inner and outer membrane, the cristae and the matrix.

Question 18

BH3-only proteins can be subdivided into activators and sensitizers. Name BH3-only proteins that are activators and name proteins that are sensitizers. What Bcl-2 proteins do these activators and sensitizers bind to?

Answer 18

• Example of activators are Bim, Bid and Puma. Activators bind and activate pro-apoptotic proteins.
• Example of sensitizers are Bad and Noxa. Sensitizers can bind and inhibit anti-apoptotic proteins.

Question 19

IAPs (Inhibitors of Apoptosis Proteins) act more downstream in the apoptotic pathway and are divided into three classes. All IAP family members contain a BIR(1, 2 or 3) domain. What is typical for class 1? And what is typical for class 2 and 3?

Answer 19

• Class 1 IAPs (mostly contain all three BIR domains and some other domains.
• Class 2 IAPs only contain the three domains (BIR1-BIR3).
• Class 3 IAPs only contain one BIR domain.

Question 20

How can apoptosis be detected in a lab? (she just summed up all these techniques, so I think it’s more important that you recognize/know these techniques than to remember all these techniques used for detection of apoptosis).

Answer 20

• Morphology (golden standard)
• DNA damage
• Viability studies
• RT-PCR, NGS
• Caspase activation (western blot, immunoprecipitation, immunohistochemistry, FACS analysis, fluorescence analysys, CRISPR-Cas)

Question 21

There are apoptosis-based therapies that have experimental strategies or novel targeted therapies. What is the focus of experimental strategies and what is the focus of novel targeted strategies?

Answer 21

• Experimental strategies stimulate apoptosis in tumor cells or inhibit anti-apoptotic blockades in tumor cells
• Novel targeted strategies make use of small molecules, peptide-mimetics, recombinant proteins, antibodies, antisense-oligomeres, viral vectors.

Question 22

How can gene translation be prevented through the use of antisense technology?

Answer 22

An antisense oligonucleotide is sequence-specific to bind and target specific mRNA. This results in the prevention of gene translation.

Question 23

What are executioner caspases (+name them)?

Answer 23

Executioner caspases get activated by caspase-8 and cleave specific protein targets for apoptosis. Caspases-3, -6 and -7 are executioner caspases.

Question 24

How is a DNA ladder generated?

Answer 24

By cleavage of DNase that cuts DNA between nucleosomes and generates a DNA ladder.

Question 25

What is the TUNEL technique?

Answer 25

A procedure used to detect apoptosis. When DNA is cleaved, the 3’ OH group is free that can be labeled with tagged nucleotides.

Question 26

Within the group of pro-apoptotic molecules is a subset referred to as the BH3-only proteins (like Bim). Why are they called BH3-only proteins?

Answer 26

Because they only share one BH domain, while most family members share three or four BH domains.

Question 27

The details of the molecular events involved in caspase-independent cell death are not known fully. However, it is known thay they also utilize proteases and facilitate permeabilization of the mitochondrial outer membrane. Name these alternative proteases.

Answer 27

Calpains, cathepsins and serine proteases. These proteases cleave target proteins to bring about morphological changes characteristic of programmed cell death.

Question 28

What is the function of Apoptosis-inducing factor (AIF)?

Answer 28

It is released from the mitochondrial intermembrane space that induces caspase-independent DNA degradation.