lecture 6 and 7 Flashcards
what is ubiquitin
a small, globular, 76 amino acid, 8.5kDa protein
where is ubiquitin found
in all eukaryotes
how many human genes express preubiquitin proteins
4, each has a diff c terminal sequence
c terminal extensions are removed to generate identical ubiquitin proteins
how is ubiquitin used as a label
when it is covalently attached to other proteins
what is ubiquitylation
tagging of substrate proteins
what acts as the receptor on the substrate protein to be ubiquitinated
the epsilon amino group of the lysine residue of the substrate protein
ubiquitin has lys 6,11,27,29,33,48,63 and terminal group met 1 which can act as acceptors of further ubiquitin donors to form chains
what is the acceptor of the c terminus of the donor ubiquitin for a linear chain
the n terminal NH2 of Met1 of ubiquitin
what linkage types do heterotypic polyubiquitin chains have
different linkage types within the same chain
how are ubiquitins extended in branched polyubiquitin chains
single ubiquitin is extended at 2 or more lys residues
how does the extended chain conformation of ubiquitin come about
linking of the c terminal carboxyl of the incoming donor ubiquitin to lys63 or met1 of the acceptor ubiquitin this positions the donor opposite the c terminal gly of the acceptor
how does a compact structure of ubiquitin chain come about
the donor ubiquitin is positioned on the side of the acceptor ubiquitin because the donor is linked to lys48 of the acceptor ubiquitin
how are different linkages of ubiquitin recognised by differed ubiquitin binding domains
some UBDs require a particular ubiquitin chain length, others dock onto the substrate as well as the attached ubiquitin
what do UBDs on regulatory proteins determine
the role and fate of the ubiquitylated protein
what does the ubd K29 determine
NFkB signalling and lysosomal degradation
what does the n terminus udb determine
dna damage responses, tnf receptor signalling and innate antiviral signalling
what does k63 ubd determine
endocytosis, NFkB signalling and dna damage responses
what does k48 ubd determine
proteosomal degradation
what is the E1 enzyme and how does it work
the ubiquitin activating enzyme
it used atp to activate the carboxyl group of ubiquitins c-terminal residue
the outcome of this reaction is the formation of a thioester between gly76 of ubiquitin and a cystine residue of the e1
- can recruit 1 of 40 e2 conjugating enzymes which accept ubiquitin
what is the e2 enzyme and how does it work
the ubiquitin conjugating enzyme
accepts the ubiquitin from e1 by a thioester linkage with a cysteine
- determine the extent and topology of the ubiquitin modification which determines the biological outcome
what is the e3 enzyme and how does it work
ubiquitin ligase enzyme
it transfers the ubiquitin onto the epsilon nh2 group of lysine on the substrate
- confer substrate specificity and RING E3 ligases catalyse the direct transfer of ubiquitin from e2 to acceptor lysine residues via an isopeptide bond
how are HECT and RING E3’s different to canonical e3s
they form a thioester intermediate with ubiquitin
what is the extra layer of regulation for polyubiquitin
polyubiquitin signals can be reversed by the action of deubiquitinating enzymes
these are particular ubds associated with the proteosome that recruit certain K48 linked proteins and deliver them as substrates for degradation
how is ubiquitin chemically activated by e1 then transferred to e2
e1 uses atp to activated terminal carboxyl group of ubiquitin - this forms a mixed anhydride with AMP that binds non-covalently to the e1
ubiquitin adenylate is then transferred to a cysteine residue resulting in a ubiquitin-E1 thiolester and amp is released
a second tightly bound ubiquitin adenylate is formed after this step
and the first ubiquitin from ubiquitin-E1 is transferred to an E2
how do e2 conjugating enzymes work
a ubiquitin conjugating domain on the e2 docks non covalently with ubiquitin-E1
the ubiquitin-E1 transfers its first ubiquitin from the cys of e1 to the e2 resulting in a thiolester bond between c terminal carboxyl of ub and the catalytic cys residue on e2
ub-e2 must dissociate from e1 so that it can interact with e3
how do hect domain e3s work
ubiquitin is transferred from the e2 to a thiol group on hect domain e3s
the ubiquitin is then conjugated to a lysine side chain of the substrate
as the chain grows the ubiquitin is attached to the core substrate
this results in the formation of an isopeptide bond between the ubiquitin and substrate
how do RING type e3 ubiquitin ligases work
RING fingers have a consensus sequence containing 8 cystine and histidine residues which coordinate 2 zinc ions in cross braced fashion
ring binds to the e2-ubiquitin and brings it closer to the substrate acceptor lys
it allosterically activates e2
as the 3 step process continues how does specificity increase
e1 interacts with all e2s which interact with a limited subset of e3s which in turn target a limited array of protein subrtrates
how does e2 determine the ubiquitin chain linkage and topology
e2 orients the acceptor ubiquitin in a way that exposes only the favoured lys residue to its active site leading to the formation of ubiquitin chains of a specific linkage
some e2s can catalyse both initiation and chain extension
others have specific roles in ubiquitin chain initiation or elongation so a different e2 is required for the priming of monoubiquitination
some e3s work with more than one e2, 1 for initiation and 1 for extension
how are substrates recognised by e3s?
substrates dock on to recognition sites (recognins) on the e3 which are distinct from hect or ring domains
a wide variety of recognins exist that dock onto diff types of motifs in the substrate that are to be selected for ubiquitylation on e3
some ubiquitin ligases have a hect domain containing a consevered cystine residue that participates in transfer of activated ubiquitin from e2 to target protein
how are the substrates recognised by e3s using the sequence motif in the substrate
E3a/Ubr1 binds to specific n terminal residues
other e3s recognise internal motifs which can sometimes be buried until they are exposed by unfolding of a protein or dissociation of a multicomplex protein
how are the substrates recognised by e3s using post translational mods of the substrate
phosphodegrons on IkBa and beta-catenin are recognised by SCFB-TrCP
SH2 domain in the E3 6bl binds phosphotyr motifs on receptors which are then ubquitylated and endocytosed
under normoxia, hydroxylated proline in transcriptor factor H1F-1 alpha is recognised by the von hipped landau component of e3 ligase
then the ubiquitylated H1F-1 alpha is degraded by a proteosome
how are the substrates recognised by e3s using post translational mods of the e3
phosphorylation of the component of the APC/cyclosome during mitosis triggers e3 to ubiquitylate
- b type cyclins which are then degraded to allow exit from mitosis
- anaphase inhibitor secretion to initiate sister chromatid separation
how are the substrates recognised by e3s via adaptor proteins
when refolding fails Hsc 70 chaperone can target misfolded proteins for ubiquitylation and degradation
in SCF how is the 50 angstrom gap between the substrate bound to the f box protein and the ubiquitin on the e2 bridged
(cullin-ring ub ligase multisubunit) the SCF is activated by covalent attachment of Nedd8 to c terminal of the cullin scaffold causes a conformational change in the cullin that allows the ring e3 (Rbx1) to swing freely
unleashed Rbx1 binds more tightly to the Ub-E2 and can reach substrates that have docked onto the fbox receptor
what are ubiquitin proteases
ubiquitin c terminal hydrolases that process ubiquitin precursor proteins to generate ubiquitn
approx 100 deubiquitylating enzymes which hydrolyse conjugated ubiquitin to regernerate monomeric ubiquitin
how does the ubiquitin-proteasome system work
e3 docks onto target proteins with an exposed degradation signal (degron)
K48 - linked ubquitin chains are added to the target
more than 4 ubiquitins in a chain are required for recognition by a proteasome and degradation
free ubiquitin is generated from processing of precursors or ubiquitin chains by UBDs
an enxymatic cascase involving e1,2 and 3 forms covalently conjugated ub chains on a target protein that are singled out by the presence of degradation signal
the ubiquitylated substrate is recognised by the proteosome where also unfolding and deubiquitylation take place prior to hydrolysis in its interior chamber
what is the proteosome
two 19s caps either side of the 20s core
19 binds to 20 whilst hydrolysing ATP
the c terminal HbVY motif on 19s AAA ATPase works like a key in the lock to induce gate opening
UBDs recognise tetra-ubiquitin on the substrate target
chaperones unfold the substrate target which is translocated through the open gate into the 20s inner chamber
the 20s core is a hollow cylinder stack of 4 heptameric rings which contain 6 proteolytic sites internally
intact ubiquitin and small peptide products are released during atp dependent process
cervical cancer caused by human papilloma virus and ubiquitin systems
human E6-AP is the eponymous HECT
e6-ap is an e3
HPV expresses e6 oncoprotein inside infected human cells
e6 binds to both p53 protein and to e6-ap
without e6, e6-ap does not normally ubiquiylate p53
maternally inherited deletion of UBE3A encoding E6-AP causes angelman syndrome
AS is characterised by intellectual and development delay, sleep disturbance, hand flapping and frequent smiling / laughing
occurs due to a loss of UBE3A gene fro maternally inherited copy of chr15
paternal silencing of UBE3A occurs in certain brain regions but only the maternal is active in cerebellum and hippocampus
ubiquitin system failure causing cancers
in certain cancers oncogenic proteins are mutated so they are no longer subject to ubiquitylation and therefore escape degradation and accumulate in the ell
other cancers involved in the over expression of e3 ligases such as mdm2 leading to enhanced degradation of p53
ubiquitin system failure causing diseases involving membrane proteins
liddles syndrom - mut in substrate docking domain of a hect e3 named Nedd4.2 prevents the docking of Nedd4.2 onto EnAC preventing ubiquitin mediated endocytosis and lysosomal degradation of the EnAC leading to hypertension through excessive sodium and water reabsorbtion
cystic fibrosis - gene encoding CFTR, a chloride ion channel, is mutated
mutated CFTR protein is ubiquitylated which marks for endocytosis and degradation so not enough CTFR reaches the cell surface
chronic neurodegenerative disease from ubiquitin system failure
alzheimers, parkinsons, dementia with lewys bodies, motor neurone diseases, huntingtons disease and frontotemporal dementias are all characterised by the occurrence of inclusion bodies on neurones containing ubiquitylated proteins
other ubiquitin like proteins
structurally similar to ubiquitin
translocated by c terminal extensions that are removed to expose invariant c terminal LRGG
each UBL has its own dedicated e1,2 and 3 enzymes
each has its own distinct although sometimes over lapping biological function
attachment may alter function, affect affinity for ligands or other entracing molecules, alter localisation or influence protein stability
example of a ubiquitin like protein (UBL)
SUMO - small ubiquitin related modifier
sumo 1,2 and 3 are mammalian forms
shares characteristics with ubiquitin
c terminal GG is essential for conjugation
linked to lysine residues in target
sumo does not have lys48 found in ubiquitin
is not directly associated with proteasomal degradation
does not make branched chain forms
sumoylation can compete with other lys modifiers
there is cross regulation between various conjugation pathways since some prtoeins can become modified by more than one ubl sometimes even at the same lysine residue
regulation of sumo by reactive O2 species
oxidise reactive rhiols on sumo enzymes
Uba1/Ass1-s-s-Ubc9
sumo can not attach to proteins if they are not sumoylated
examples of sumo functions (RanGAP1, IkB, c-JUn, p53 and mdm2)
RanGAP1 - causes nuclear translocation
IkB - blocks Ub conjugation sites, prevents degradation
c-Jun - inhibits transcriptional activity
p53 and mdm2 - blocks mdm2 self ubiquitination, prevents degradation and sumo-p53 in dna binding domain decreases apoptotic activity
