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what are PTM writers?
molecules which add chemical changes to a protein
what are PTM editors?
molecules which remove the PTM from a molecule
what are readers?
molecules which can induce changes after recognising the biochemical changes
give some responses which can be produced by readers
localisation, enzyme activity, complex function and stability changes
what are the writers and editors in phosphorylation?
writers are kinases
editors are phosphotases
what are readers in phosphorylation?
phosphopeptide binding domains
what size modification are most PTM? what size are the modifications in the red box?
normally small molecules
in the red box are whole protein complex modifications (additions of protein complexes)
what residues do UB and SUMO bind to?
lysine
how large is the UB and SUMO complexes?
UB is 9 Kda
SUMO is 11KDA
pretty large relative to the molecules theyre being added to
compare UB and SUMO structrually
they look pretty similar in their crystal structure
compare the amino acid structures between SUMO and UB
very different in amino acid structrue - only 9 amino acids shared between the 2
compare SUMO1 to SUMO2
both are similar but still destinct from each other
list the 3 enzymes needed to add ubiquitin to a complex
E1 activating enzymes
E2 conjugating enzymes
E3 ligases
which of the 3 E enzymes involved in ubiquitination performs the energy dependant step?
the E1 activating enzyme
what does the E1 activating enzyme do?
binds itself to free Ub using ATP
after the E1 enzyme is bound to UB what how does it interact with the E2 conjugating enzyme?
the UB is transfered from the E1 to the E2 enzyme
is transfer of UB from E1 to E2 energy dependant? explain
no - because the same number of bonds are broken as those made
once the E2 conjugating enzyme has the UB what happens?
it associates with the E3 ligase to form a E2-E3 complex
what does the E2-E3 complex do?
adds Ub to the substrate
can E2 hand Ub straight to E3?
in some cases yes but not as common
what is the name of the enzyme which removes ubiquitin?
deubiquitinase
how many E1 proteins exist?
only 2
how many E2 proteins exist?
around 40
how many E3 proteins exist?
around 700
can multiple E3's work on a single E2?
yes
how specific is the ubiquitin pathway?
highly specific
what is the purpose of having the UB cascade laid out like this?
to amplify the signal
what is multi-monoubiquitination
when a single molecule has multiple Ub's bound on different lysine molecules
why is it significant that UB itself had multiple lysine residues?
its means the Ub molecule can be ubiquitinated itself
how many lysines are present on UB?
7 different types
how many Ub's can be added to Ub and why?
can have 8 added to a single UB
1 to each of the 7 lysine residues and 1 to the N terminus chain
how can there be many different types of di-ubiquitination?
because Ub can be added to the different lysine residues in many different combinations
what is the K48 di-UB chain involved in?
involved in proyeosomal degredation
how many famlies of UB readers are there?
over 20
what is RAP80
a UB reader protein used for blood complexes in DNA damage
what does UIM stand for in terms of ubiquitin readers?
"Ubiquitin Interacting Motifs" - regions of UB readers which interact with UB
how does RAP80 measure poly-UB?
acts as a ruler to measure how far apart the UB molecules are to see if its the correct UB for its function
how specific is the RAP80 reading process?
very specific
what is the function of RNF168?
is a E3 ligase which adds K63 linked ubiquitin chains to histones
how does the RNF168 Ub reader self regulate?
is reads the level of K63 linked ubiquitination in its surroundings to modulate its activity
specifically what type of ubiqitination does RNF168 recognise?
ubiquitination of K63 which means the 63rd lysine residue of its target molecule
can you have multiple UB reading domains on the same protein?
yes
what levels of the E protein UB cascade give the specificity and flexability?
the E2-E3 complex gives flaxability
while the presence of multiple E3's per E2 provides specificity to the target protein
will all proteins have UB added to it in its life?
all proteins will likely have UB but not all are degraded
just a heads up:
DNA occurs in chromatin which isnt naked - the nucelosomes are wrapped by DNA
commonly DNA and histones have lots of PTM's
just heads up for the rest of the flash cards to keep this in mind because the diagrams will probs show linear DNA but thats not true
when a DSB occurs what happens to MRE11 to activate it?
it is phosphorylated
give some functions of the MRN complex
to recruit and provide a binding site for ATM
to act as a scaffold
to tether the 2 broken DNA segments together
what does ATM do to the histone during DSB?
phosphorylates the histone tail to give gamma H2AX from H2AX
what UB writer recruiter is recruited by gamma H2AX? what type of PTM recruits this protein
MDC1 recruited by phosphorylation
what does MDC1 recruit?
RNF8
when does MDC1 recruit RNF8?
when ATM phosphorylates H2AX
what type of protein is RNF8?
an ubiquitin writer
what does RNF8 stand for?
Ring Finger Protein 8 (aparently)
what type of UB modification is added by RNF8 and to which histone is it added?
RNF8 adds K63 chains to the histone H1 protien
what is the purpose of H1 modification?
targets the replication proteins for degredation which arent needed during the DNA damage response
what does RNF8 do to JMJD2A?
adds K48 UB chains marking it for proteosome degredation
what type of protein is JMJD2A?
a histone demethylase enzyme
(aparently... even tho laura said they dont exist)
what does RNF168 do in the DNA damage response
reads the RFN8 induced K63 UB signal and amplifies it
what is the purpose of amplifying the RNF8 signal?
to show where DNA damage is
what motif does RNF8 use to read K63 linked UB signals?
the UIM
other than amplifying the K63 signal what does RFN168 do?
puts down dedecated monomodification on H2A
what is the outcome of the K63 Ubiquitination in the DNA damage response?
recruits BRCA1 and 53BP1 complexes
what are the 2 main methods of DSB repair?
HR or NHEJ
what protein cross talk determiens if HR or NHEJ is performed?
cross talk between BRCA1 and 53BP1
which out of BRCA1 and 53BP1 is dominant in G1?
53BP1
what is 53 PB1?
a ubiquitin reader
what does 53BP1 read?
the H2a-K13 mark and H2K20Me3
does resection of the 5' strand after DSB lead to activation of NHEJ or HR?
HR - NHEJ is inhibited by rescection because it cant recognise single strand overhangs
what does 53BP1 do to BRCA1?
blocks its activity
what is the outcome of 53PB1 blocking BRCA1?
means you dont get rescection of the broken DNA meaning you switch to NHEJ rather than HR
how does 53BP1 inhibit rescection?
it inhibits the exonucelase enzymes involved in rescection
why can you not perform NHEJ after resceting the 5' end of DNA?
(other than because NHEJ machinery cant recognise single strand overhangs)
because the rescected DNA will be lost as there is no homologous template to repair the DNA
what is dominant out of BRCA1 and 53BP1 in S and G2 phase?
BRCA1 is dominant
is BRCA1 linked to HR or NHEJ?
HR
does BCRA1 have ubiquitin ligase activity?
yes
which histone does BRCA1 lay down markers onto?
onto the H2A termins
what reads the markers laid down by BRCA1?
SMARCAD1
what does SMARCAD1 do?
remodels the nucelosome and moves 53BP1 away from the damage site
what else is promoted by BCRA1?
(HINT: this prevents NHEJ)
recruitment of rescection enzymes to rescet the 5' strand leaving a 3' overhang
give examples of rescection enzymes recruited by BRCA1
CTIP, BLM, Exo1
what syndrome is associated with mutations in RF168?
RIDDLE syndrome
what disease is BRCA1 mutation liked to?
breast and ovarian cancer + fanconi anemia
why does BRCA1 mutation cause high levels of genomic instability?
causes a loss of HR
what pathway is used by the cell if it loses HR in S and G2 phases?
the PARP pathway
what treatment is initially responsive in cancer cells using the PARP pathway
PARP inhibitors - because the cells cant process PARPi mediated DNA damage so the cell dies
how does the cancer cell restore HR?
loses 53PB1 so the cell starts using HR again...
what enzyme removes SUMO?
SUMO protease enzymes
how many E molecules are present in SUMO?
3 E molecules (E1,E2 and E3)
how many SUMO E1 molecules exist?
1
how many SUMO E2 molecules exist?
1
how many SUMO E3 ligases exist?
12
specifically how does SUMO addition work?
very very similar to UB
what residue is SUMO added to?
lysine
how many isoforms of SUMO are there?
3
which 2 SUMO molecules are almost identical?
2 and 3
can you get poly SUMO?
you can on SUMO 2 and 3
but cant get poly SUMO on SUMO1
what motifs are present on SUMO readers?
SUMO interacting motifs
what differentiates different forms of SUMO
the different orientations of SUMO on the target protein
give some ways in which SUMO involved in the DNA damage response?
recruits complexes (this is all that was written on the slide)
activates BRCA1 ligase
regulates chromatin state
* P.s no idea what complexes are recruited
name 2 SUMO E3 ligases
PIAS1 and PIAS4
what Ubiquitin protein recognises polySUMO for cross talk?
RNF4
what does RFN4 do once its recognised polySUMO?
adds UB onto the SUMO chains
what is the outcome of UB-SUMO mixed chains in the DNA damage response?
causes protein degredation which aids in the timing of the DNA damage response
what proteins are degraded by UB-SUMO hybrid chain labelling?
DNA scafflod proteins
what does SENP2 do?
limits SUMO chain on MDC1
whats the purpose of SENP2 limiting SUMO on MDC1?
restructs MDC1 Ubiquitination and removal from sites of DNA damage
- so it controls the growth rate of UB chains on MDC1
here is a summary
:)
good luck... this lecture is awful i have no idea whats going on... so many proteins and so many random things to remember
:(