L4 - PTMs Flashcards
what is a PTM?
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
what do PTM give you?
- information transfer
- signal amplification
- cross-talk between pathways
what can PTMs do to a protein?
- 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
what is phosphorylation?
addition of a phosphate to either a tyrosine, threonine or serine
adds a negative charge
how does phosphorylation occur?
gamma phosphate group of ATP donates the phosphate to the base by nucleophilic attack
what bases can phosphorylation occur at?
- tyrosine
- threonine
- serine
how many human protein kinases are there?
518
why is phosphorylation/dephosphorylation an important control mechanism?
- it is rapid
- doesn’t require new proteins to be made/degraded
- easily reversible
why does lysine get modified in lots of different ways?
has a very reactive positive epsilon-amino group
what is lysine acetylation?
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
how do acetylases transfer the acetyl group to the epsilon-amino group?
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
what are HATs?
histone acetyltransferases
co-activators that activate gene transcription
what are HDACs?
histone deacetylases
co-repressors that cause gene silencing
lysine methylation
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
arginine methylation
has a positively charged side chain with an amino group
comes in the de-methylated form
involved in chromatin structure
what histone modifications control chromatin structure?
lysine acetylation
lysine methylation
arginine methylation
what are writers?
- proteins that deposit the modification
- write the modification code
- kinases, acetylases, methylases
what are readers?
- proteins that recognise modifications
* read and translate the code
what are erasers?
proteins that remove modifications
what do the different protein domains do?
can recognise phosphorylated motifs in target proteins
what does the SH2 domain recognise?
recognises phospho-tyrosine in cell surface receptors
• allows the docking of proteins that promote growth and survival
what does the WW domain recognise?
recognises phospho-serine/proline
• helps cell cycle control
what does the FHA domain recognise?
recognises phospho-threonine domains
• is a DNA damage checkpoint
what does the family of 14-3-3 proteins recognise?
recognises distinct phospho-serine/threonine motifs
• important in cytosolic retention and regulation
what is BD?
bromodomain
what is CD?
chromodomain
combinational PTMs
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
what are histones?
proteins that condense and structure the DNA of eukaryotic cell nuclei into units called nucleosomes
they impact gene regulation - positively and negatively
what are the core histones?
H2A
H2B
H3
H4
what is the nucleosome core formed of?
2 H2A-H2B dimers
1 H3-H4 tetramer
what are the histone H3 variants and modifications?
histone 3K4
histone 3K9
histone 3K27
(4,9,27)
what are the histone H4 variants and modifications?
histone 4K5 histone 4K8 histone 4K12 histone 4K16 histone 4K20
(5,8,12,16,20)
histone 3K4
activation
can be methylated and acetylated
histone 3K9
turns genes on when acetylated
silences genes when methylated
histone 3K27
shuts down transcription
trimethylation is associated with inactive gene promoters
acts in opposition to H3K4me3
histone 4K5
closest lysine residue to N-terminal tail of H4
can be acetylated and methylated
activator
histone 4K8
lysine on tail of H4
only acetylated
activator
histone 4K12
lysine on tail of H4
only acetylated
activator
part of a ‘backbone’ of histone modifications that are associated with active promoters
histone 4K16
lysine on tail of H4
only acetylated
linked with both transcriptional activation and repression
histone 4K20
lysine on tail of H4
only methylated - mono, di or tri
mono and di methylation associated with activation
tri methylation associated with repression
how is H4 different to H3?
it has less sequence variation
structurally restrained by evolution
how can we detect some PTMs?
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
how can we find the locations of modifications?
by creating modification specific antibodies
mass spectroscopy can also be used
how do we create modification specific antibodies?
- immunise animal with peptide and take blood and serum from the animal
- take the mixture of antibodies present in the serum and put them down a fractionation column composed of phosphopeptide
- when column is eluted, antibodies that specifically recognise the phosphopeptide are left in the column
- this will still be an antibody mix as contains antibodies that recognise the non-phosphorylated peptide
- a column made of non-phosphorylated peptide and antibody mixture poured in
- antibodies specific for phosphorylation flow through
left with antibodies that only recognise the phosphorylated form of the protein
