define: tumor suppressor gene

what happens when the tumor suppressor gene is mutated?

when this is functioning (active), it stops the formation of tumors

when tumor suppressor genes are mutated, their activity is not able to stop tumors and therefore cause cancers

False:

not necessarily

however yes, most tumors are due to mutations on the p53 gene

*50% of all tumor types in humans are due to mutations on the p53 tumor supressor gene*

cell cycle arrest

and

apoptosis

True:

p53 is not essential for normal functioning of cells

T/F: p21 has earned the nickname "guardian of the genome"

Why?

False:

p53 has earned that nickname b/c it maintains the genomic integrity (cell cycle arrest, apoptosis, DNA repair)

first, p53 will try to repair the mutation

if that doens't work, then p53 will undergo apoptosis

it means that if there is too much p53 protein in the cell, the cell will get rid of some of it so the p53 is not taking over the cell: this literally means post translational regulation

in the online notes, it says "accumulation of active p53 in response to stress occurs mainly through post translational mechanisms" - this is what I wrote above

steps:

1) cellular stress

2) induction of p53 gene

3) p53 proteins produced

4) assuming no regulation at transcriptional level which leads to too much p53 protein produced

5) cell gets rid of some of p53 proteins

6) known as post translational mechanism

True:

p53 is a gene-specific transcriptional activator which relies on its ability to bind defined sequence elements within target genes

negative regulation

the sequence-specific DNA binding activity is subject to constitutive negative regulation

1) relieving the inhibition of its C-terminal resulting in increased DNA binding

2) modifications of the N-terminal transactivation domain enabling a much more efficient recruitment of components of the transcription machinery

3) change in subcellular localization: latent p53 may be in the cytosol while active p53 may be in nucleus

Mdm2 protein* (not gene)

Mdm2 protein is the product of an oncogene

1) inactivation of p53 by not allowing transcription

2) complete elimination of p53 by proteolytic degradation

both of these are done by binding of mdm2 with p53

p53 protein binds to mdm2 gene* (not protein)

recall that mdm2 protein* binds to p53 gene* in order to inactivate the p53 gene

False:

p53 is killed (proteolytic degradation) when mdm2 protein binds to p53 protein

point of asking this question: we know p53 can be inactivated or killed and both happen when mdm2 protein binds to p53 protein

the only thing that happens when p53 protein binds to mdm2 gene is the stimulation of mdm2 gene to make mdm2 proteins

when p53 protein binds to mdm2 gene to stimulate its transcription is positive feedback

when mdm2 proteins are produced in response to the positive feedback stated above and the mdm2 protein binds to p53 protein and inactivates or kills p53 protein is negative feedback

1) non-functional mdm2 gene: embryonic lethality

2) non-functional mdm2 protein: deregulation of p53 to an extent that its activity leads to embryonic death

3) excessive mdm2 gene: constitutive inhibition of p53 and thereby promoting cancer without a need to alter the p53 gene itself

False:

we know that excessive mdm2 gene leads to constitutive inhibition of p53 and thereby promoting cancer without the need to alter the p53 gene itself.

excessive mdm2 gene can also promote cancer independently of p53*

1) its a nuclear phosphoprotein that binds to and inhibits transactivation by tumor protein p53 as part of an autoregulatory negative feedback loop

2) the protein has E3 ubiquitin ligase activity which targets tumor protein p53 for proteosmal degradation

3) protien affects cell cycle, apoptosis, and tumorigenesis through interactions with other proteins

True:

E3 ubiquitin ligase activity is found on mdm2 protein and when it binds to p53 protein, causes the p53 protein to undergo proteolytic degradation

activation of p53 is primarily achieved through _______

demise of p53 is primarily achieved through _______

p53 stabilization

ubiquitin-proteasome pathway

are primarily due to continuous mdm2 promoted degradation

binding of mdm2 protein and p53 protein

covalent modification of p53 generally means what?

what is the result?

means that the p53 protein* has been phosphorylated

the result is that it causes stabilization and activation of p53 through inhibition of p53 ubiquitination and degradation - in other words, phosphorylating p53 protein prevents degradation by mdm2 protein

False:

yes, the phosphorylation occurs on the transactivation domain of the p53 protein but phosphorylation can occur on either mdm2 protein or p53 protein

phosphorylating either binding domains inhibits binding of the two proteins thereby preventing degradation of p53

True:

specific amino acids on the binding domain of p53 are phosphorylated in order to bring specific outcomes

for ex: phosphrylation of specific amino acid on p53 is necessary for repair of ionization radiation and some other types of DNA damage

False:

that is one possibility but phosphorylation may actually retain binding ability of p53 with mdm2 domain but may impair the proteolytic ability of mdm2 protein

what happens when binding domains of both the mdm2 protein and the p53 protein are phospohrylated?

True:

ARF protein can bind to mdm2 protein and p53 protein but it binds to mdm2 protein more commonly

recall that phosphorylation can occur on either mdm2 binding domain or p53 binding domain and its the same with ARF protein however it binds to mdm2 protein more than it binds to p53 protein

ARF is a regulatory protein that binds to mdm2 protein

binding of ARF to mdm2 protein leads to stabilization of p53 b/c although binding of p53 and mdm2 protein occurs, it does not allow for ubiquitination and therefore degradation

ARF arises through translation of an alternative reading frame derived from INK4A (aka p16 gene)

INK4A's (p16 gene) product is a suppressor of CDK4A

define: cyclin dependent kinase

define: cyclin

cyclin depenent kinases are protein kinases that regulate cell cycle progression in all eukaryotes and require physical association with cyclins to achieve full enzymatic activity

cyclin are proteins that bind to CDKs helping them in cell maturation

well we already that covalent modification (phosphorylation) of either mdm2 protein or p53 protein can inhibiting binding of the two proteins thereby preventing ubiquitination and degradation of p53 so the p53 can remain stabilized and continue functioning

another way that we have already mentioned is ARF protein which binds to mdm2 protein (and p53 protein to a lesser extent) which leads to the binding of the two proteins but does not allow ubiquitination and degradation of p53 thereby allowing the p53 to remain stabilized and continue functioning

the p53 protein has a hydrophobic element which allows for the p53 protein to get into the nucleus

this p53 protein that is entering the nucleus is known as a nuclear phosphoprotein

induction of ARF protein

preventing ubiquitination and degradation of p53 protein

1) UV is known to cause distinct mutations in keratinocytes

2) keratinocytes = epidermal cells that make keratin

3) keratin = protein important in hair, skin, nails and ultimately contributes to development of non-melanoma skin cancers such as basal cell carcinoma and squamous cell carcinoma

4) process by which these mutations are introduce into these cells begin with the interaction between UV photons and cell DNA

5) as photons are absorbed by DNA, an excited state is produced rearranging electrons and producing a photoproduct

6) photoproducts if not repaired interfere with DNA replication and cause specific mutations in DNA

7) mutations caused by UV-B in DNA cause a single base shift of C by T

8) mutations caused by UV-A in DNA are not well characterized -- UV-A mutations are known to be 1000 times less mutagenic than UV-B

9) molecular basis of nonherited melanomca are not completely understood

10) sunlight plays a role

proto-oncogenes

and

tumor suppressor genes

so either of these genes can have their DNA damaged due to exposure to UV radiation

however, mutations in the tumor suppressor genes are thought to play a critical role in precancerous lesions and have been in implicated in all types of skin cancers

on the N-terminal (amino side): we have the transactivation domain that binds to mdm2 protein, then going left to right we have the DNA binding domain for transcription, then TET (tetramerization domain and on the C-terminal) which contains NES and NLS - the NES is for p53 going into the nucleus and the NLS is for p53 to find the gene, then at last, we have the NEG which binds to damaged DNA and this is also the place where ubiquitin binds

note: Mdm2 binds on the N-terminal while ubiquitin binds on the C-terminal

first, the NES allows p53 to enter the nucleus, then NLS allows p53 to find the target gene which it will transcribe, and lastly the DNA binding domain will allow p53 to bind to that target gene

False:

TET consists of NES and NLS

NEG is what allows p53 to bind to damaged DNA and this is where ubiquitin binds

TET domain

what we mean by making the p53 protien biologically functional is that the binding of the target gene is located on the TET domain therefore the TET domain is the one that is responsible for making the p53 protien functional

False:

the DNA binding domain which is in the middle of the p53 protein is not where the target gene binds

this is the location where the gene binds for transcription

the location where the target gene binds is on the TET domain

what is the target gene that the p53 protein binds to?

where does the target gene bind on the p53 protein?

the damaged DNA is the target gene that p53 protein binds to

the damaged DNA binds on the TET domain of the p53 protein

DNA-binding domain

95% of the mutations occur on this domain

tetramer

1 monomer p53 + 1 monomer p53 = 1 dimer p53

1 dimer p53 + 1 dimer p53 = 1 tetramer p53

True:

dimers or monomers are not biologically functional

_______ base pairs make up one full spiral of DNA

________ angstrom between two base pairs

length of entire spiral = _______ angstrom

10 base pairs

3.4 angstrom between two base pairs

34 angstrom = length of entire spiral

False:

each dimer* (not monomer) binds to 10 base pairs

20 base pairs

b/c one dimer binds to 10 base pairs so one tetramer binds to 20 base pairs

what has current study about the location of mutations on p53 dimer other than the fact that mutations are located on DNA binding domain on p53?

what is the importance of this?

the point of contact between the two core domains of a pair of p53 forming a dimer is related to the part of the protein where the mutation occurs

so when two monomers p53 bind each other to form a dimer, the point of contact where they bind is related to the part of p53 where the mutation occurs

this is imporant b/c this suggests that this point of contact is as important as the interface between tetramer binding to DNA b/c the interface of tetramer binding to DNA is dependent on where the point of contact where the two monomers bind

p53 binds to the downstream region of the damaged gene

it binds here b/c downstream region is responsible for growth so binding and activating expression of this region inhibits growth of the damaged DNA

these are enzymes that regulate the cell cycle like the checkpoints of cell cycle

CDK = cyclin dependent kinases

what is CDK2, CDK4, and CDK6?

what happens if these two kinases are disrupted?

these are kinases that regulate the G1 phase

if these two kinases are disrupted, the G1 phase of the cell cycle is rested

what are the stages of the cell cycle?

how many checkpoints are there in the cell cycle?

when do the checkpoints occur?

we have G1 phase = growth and development

S phase = DNA replication

G2 phase = growth and development

M phase = cell division

there are 3 checkpoints

first checkpoint = between G1 and S phase

second checkpoint = between S and G2 phase

third checkpoint = between G2 and M phase

the DNA binding domain as we know is the region where DNA binds to p53

we know that this DNA is not DNA of the target gene

this DNA is of some gene (i dont know what in particular) but binding to this DNA will in turn activate p21 gene to make p21 protein and this p21 protein will interact with CDK and this complex of p21 and CDK will cause cell cycle arrest so the p53 protein can repair whatever damage that has occurred on the damaged DNA

True:

there are no checkpoints available in cancer cells

so this allows cancer cells to become immortal (keep dividing) b/c there is no regulation of the cell cycle

cyclin E + CDK2

cyclin D + CDK4

cyclin D + CDK6

1) damaged DNA activates p53 = transcriptional activator of p21

2) p21 gene into p21 proteins

3) inhibits all the cyclins (cyclin D and E) from binding to their respective CDKs (CDK 2,4,6) so CDK cannot function and causes cell cycle arrest

4) G1 phase cannot proceed to S phase anymore

5) given enough time of this cell cycle arrest, DNA damage repair will occur

False:

first of all, CDK are not bound to p53

CDK can only function when they are bound to cyclin** hence named cyclin-dependent kinases

CDKs phosphorylate cell cycle regulatory proteins

we know that CDK/cyclin complexes are important for cell cycle progression and we know kinases (like CDK) phosphorylate things so CDK will of course phosphorylate proteins that are cell cycle regulatory proteins

we know that CDKs are kinases meaning that they phosphorylate things

well it turns out that CDKs in particular phoshporylate amino acid residues serine and threonine therefore CDKs are called serine/threonine kinases

it depends on what kind of a kinase the CDK is bound to

for example: if CDK2,4,6 are bound to cyclin D and E, then the CDK/cyclin complex regulate G1/S checkpoint of the cell cycle

which gene binds to cyclin D?

which gene binds to cyclin E?

which gene binds to cyclin A?

p21, p27, p57, and p16 bind to cyclin D and E

p21 and p27 bind to cyclin A

yes and no

cell cycle arrest does mean that the cell cycle stops at a particular phase (wherever the cell cycle arrest took place) BUT this is only for a given period of time - in other words, once that time is over, the cell cycle will continue progression and go on

there are 5 things:

p21, p27, p57, p16, and cyclin G1

which inhibitors are part of the Cip family?

which are part of the Kip family?

p21 is part of the Cip family

p27, p57, and p16 are part of the Kip family

p21, p27, p57, and p16

dunno this one

kip1 = p27

kip2 = p57

WAF = p21

CDK4i = p16 (i = inhibitor)

recall that these two proteins are anti-apoptotic proteins meaning they will tell the cell to not die

in order to make them non-functional (ie. so the cell does die), PUMA protein has to bind to them

1) PUMA protein beinds to Bcl-2 and Bcl-xL making these two proteins non-functional causing mitochondrial membrane permeabilization

2) PUMA protein causing p53 to be removed from Bcl-xL so p53 can activate Bax and induce mitochondrial membrane permeabilization

False:

PUMA protein causes p53 to be removed from Bcl-xL protein not Bcl-2 protein

how does p53 function when PUMA protien mediates apoptosis induced by nuclear p53?

how does p53 function when PUMA protein mediates apoptosis induced by cytoplasmic p53?

1) nuclear p53 - p53 functions as transcriptional activator making PUMA proteins which can bind to Bcl-2 and Bcl-xL and making them non-functional

2) cytoplasmic p53 - PUMA displaces cytoplasmic p53 from Bcl-xL, allowing p53 to activate Bax and cause cell death

we do this by taking a small molecule antagonist of Mdm2**

we know that mdm2 has a p53 binding domain which is the place where p53 binds and this binding causes mdm2 to inactivate or kill p53 (proteolytic degradation)

but if p53 is inactivated or killed then p53 cannot repair or kill cancer cells - we want lots of p53 in the body

so in in vivo activation of p53, just so p53 cannot bind to mdm2 protein, we place another antagonist so that way, p53 survives and can kill cancer cells

what are nutlins?

True:

nutlins bind to the p53 binding domain on mdm2 proteins