L4 - PTMs

Question 1

what is a PTM?

Answer 1

the addition of a chemical group or molecule to specific amino acids of a protein

can generally be added or removed

add to the complexity of the proteome

Question 2

what do PTM give you?

Answer 2

• information transfer
• signal amplification
• cross-talk between pathways

Question 3

what can PTMs do to a protein?

Answer 3

• create/block a binding site
• change the conformation of a protein
• affect stability of protein
• affect location of protein in cell
• lead to rapid amplification
• allow cross-talk between pathways

Question 4

what is phosphorylation?

Answer 4

addition of a phosphate to either a tyrosine, threonine or serine

adds a negative charge

Question 5

how does phosphorylation occur?

Answer 5

gamma phosphate group of ATP donates the phosphate to the base by nucleophilic attack

Question 6

what bases can phosphorylation occur at?

Answer 6

• tyrosine
• threonine
• serine

Question 7

how many human protein kinases are there?

Answer 7

518

Question 8

why is phosphorylation/dephosphorylation an important control mechanism?

Answer 8

• it is rapid
• doesn’t require new proteins to be made/degraded
• easily reversible

Question 9

why does lysine get modified in lots of different ways?

Answer 9

has a very reactive positive epsilon-amino group

Question 10

what is lysine acetylation?

Answer 10

the addition of an acetyl group by acetyl-CoA by acetylases

neutralises positively charged epsilon-amino group and creates a binding site for specific proteins that recognise acetylated lysine

Question 11

how do acetylases transfer the acetyl group to the epsilon-amino group?

Answer 11

a conserved glutamate residue in the acetylase activates a water molecule for removal of a proton from the amine group on lysine
activates it for nucleophilic attack on the carbonyl carbon of enzyme bound acetyl-CoA

Question 12

what are HATs?

Answer 12

histone acetyltransferases

co-activators that activate gene transcription

Question 13

what are HDACs?

Answer 13

histone deacetylases

co-repressors that cause gene silencing

Question 14

lysine methylation

Answer 14

doesn’t necessarily neutralise the positive charged epsilon-amino group

comes in 3 different types - mono, di and tri
• recognised by different proteins
• increases diversity of modification

S-Adenosyl methionine (SAM) serves as a co-factor & methyl donor group for lysine (and arginine) methylation

Question 15

arginine methylation

Answer 15

has a positively charged side chain with an amino group

comes in the de-methylated form

involved in chromatin structure

Question 16

what histone modifications control chromatin structure?

Answer 16

lysine acetylation
lysine methylation
arginine methylation

Question 17

what are writers?

Answer 17

• proteins that deposit the modification
• write the modification code
• kinases, acetylases, methylases

Question 18

what are readers?

Answer 18

• proteins that recognise modifications

* read and translate the code

Question 19

what are erasers?

Answer 19

proteins that remove modifications

Question 20

what do the different protein domains do?

Answer 20

can recognise phosphorylated motifs in target proteins

Question 21

what does the SH2 domain recognise?

Answer 21

recognises phospho-tyrosine in cell surface receptors

• allows the docking of proteins that promote growth and survival

Question 22

what does the WW domain recognise?

Answer 22

recognises phospho-serine/proline

• helps cell cycle control

Question 23

what does the FHA domain recognise?

Answer 23

recognises phospho-threonine domains

• is a DNA damage checkpoint

Question 24

what does the family of 14-3-3 proteins recognise?

Answer 24

recognises distinct phospho-serine/threonine motifs

• important in cytosolic retention and regulation

Question 25

what is BD?

Answer 25

bromodomain

Question 26

what is CD?

Answer 26

chromodomain

Question 27

combinational PTMs

Answer 27

modifications such as methylation, phosphorylation or acetylation are commonly recognised by proteins with PTM-recognition sites modifications such as lysine methylation can occur up to 3 times on a single residue resulting in PTMs with distinct activity neighbouring PTMs have different affects on the ability of proteins to recognise a phosphorylation site they determine which protein-protein interactions lead to distinct biological outcomes

Question 28

what are histones?

Answer 28

proteins that condense and structure the DNA of eukaryotic cell nuclei into units called nucleosomes they impact gene regulation - positively and negatively

Question 29

what are the core histones?

Answer 29

H2A H2B H3 H4

Question 30

what is the nucleosome core formed of?

Answer 30

2 H2A-H2B dimers | 1 H3-H4 tetramer

Question 31

what are the histone H3 variants and modifications?

Answer 31

histone 3K4 histone 3K9 histone 3K27 (4,9,27)

Question 32

what are the histone H4 variants and modifications?

Answer 32

``` histone 4K5 histone 4K8 histone 4K12 histone 4K16 histone 4K20 ``` (5,8,12,16,20)

Question 33

histone 3K4

Answer 33

activation can be methylated and acetylated

Question 34

histone 3K9

Answer 34

turns genes on when acetylated silences genes when methylated

Question 35

histone 3K27

Answer 35

shuts down transcription trimethylation is associated with inactive gene promoters acts in opposition to H3K4me3

Question 36

histone 4K5

Answer 36

closest lysine residue to N-terminal tail of H4 can be acetylated and methylated activator

Question 37

histone 4K8

Answer 37

lysine on tail of H4 only acetylated activator

Question 38

histone 4K12

Answer 38

lysine on tail of H4 only acetylated activator part of a 'backbone' of histone modifications that are associated with active promoters

Question 39

histone 4K16

Answer 39

lysine on tail of H4 only acetylated linked with both transcriptional activation and repression

Question 40

histone 4K20

Answer 40

lysine on tail of H4 only methylated - mono, di or tri mono and di methylation associated with activation tri methylation associated with repression

Question 41

how is H4 different to H3?

Answer 41

it has less sequence variation structurally restrained by evolution

Question 42

how can we detect some PTMs?

Answer 42

some modifications can be visualised since they change the mobility of a protein on SDS-PAGE the negative charge introduced by phosphorylation displaces negatively charged SDS from the protein, causing it to migrate more slowly in the gel however, this gives no information about the locations of the modifications

Question 43

how can we find the locations of modifications?

Answer 43

by creating modification specific antibodies mass spectroscopy can also be used

Question 44

how do we create modification specific antibodies?

Answer 44

1. immunise animal with peptide and take blood and serum from the animal 2. take the mixture of antibodies present in the serum and put them down a fractionation column composed of phosphopeptide 3. when column is eluted, antibodies that specifically recognise the phosphopeptide are left in the column 4. this will still be an antibody mix as contains antibodies that recognise the non-phosphorylated peptide 5. a column made of non-phosphorylated peptide and antibody mixture poured in 6. antibodies specific for phosphorylation flow through left with antibodies that only recognise the phosphorylated form of the protein