lecture 6 and 7

Question 1

what is ubiquitin

Answer 1

a small, globular, 76 amino acid, 8.5kDa protein

Question 2

where is ubiquitin found

Answer 2

in all eukaryotes

Question 3

how many human genes express preubiquitin proteins

Answer 3

4, each has a diff c terminal sequence

c terminal extensions are removed to generate identical ubiquitin proteins

Question 4

how is ubiquitin used as a label

Answer 4

when it is covalently attached to other proteins

Question 5

what is ubiquitylation

Answer 5

tagging of substrate proteins

Question 6

what acts as the receptor on the substrate protein to be ubiquitinated

Answer 6

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

Question 7

what is the acceptor of the c terminus of the donor ubiquitin for a linear chain

Answer 7

the n terminal NH2 of Met1 of ubiquitin

Question 8

what linkage types do heterotypic polyubiquitin chains have

Answer 8

different linkage types within the same chain

Question 9

how are ubiquitins extended in branched polyubiquitin chains

Answer 9

single ubiquitin is extended at 2 or more lys residues

Question 10

how does the extended chain conformation of ubiquitin come about

Answer 10

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

Question 11

how does a compact structure of ubiquitin chain come about

Answer 11

the donor ubiquitin is positioned on the side of the acceptor ubiquitin because the donor is linked to lys48 of the acceptor ubiquitin

Question 12

how are different linkages of ubiquitin recognised by differed ubiquitin binding domains

Answer 12

some UBDs require a particular ubiquitin chain length, others dock onto the substrate as well as the attached ubiquitin

Question 13

what do UBDs on regulatory proteins determine

Answer 13

the role and fate of the ubiquitylated protein

Question 14

what does the ubd K29 determine

Answer 14

NFkB signalling and lysosomal degradation

Question 15

what does the n terminus udb determine

Answer 15

dna damage responses, tnf receptor signalling and innate antiviral signalling

Question 16

what does k63 ubd determine

Answer 16

endocytosis, NFkB signalling and dna damage responses

Question 17

what does k48 ubd determine

Answer 17

proteosomal degradation

Question 18

what is the E1 enzyme and how does it work

Answer 18

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

Question 19

what is the e2 enzyme and how does it work

Answer 19

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

Question 20

what is the e3 enzyme and how does it work

Answer 20

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

Question 21

how are HECT and RING E3’s different to canonical e3s

Answer 21

they form a thioester intermediate with ubiquitin

Question 22

what is the extra layer of regulation for polyubiquitin

Answer 22

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

Question 23

how is ubiquitin chemically activated by e1 then transferred to e2

Answer 23

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

Question 24

how do e2 conjugating enzymes work

Answer 24

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

Question 25

how do hect domain e3s work

Answer 25

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

Question 26

how do RING type e3 ubiquitin ligases work

Answer 26

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

Question 27

as the 3 step process continues how does specificity increase

Answer 27

e1 interacts with all e2s which interact with a limited subset of e3s which in turn target a limited array of protein subrtrates

Question 28

how does e2 determine the ubiquitin chain linkage and topology

Answer 28

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

Question 29

how are substrates recognised by e3s?

Answer 29

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

Question 30

how are the substrates recognised by e3s using the sequence motif in the substrate

Answer 30

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

Question 31

how are the substrates recognised by e3s using post translational mods of the substrate

Answer 31

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

Question 32

how are the substrates recognised by e3s using post translational mods of the e3

Answer 32

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

Question 33

how are the substrates recognised by e3s via adaptor proteins

Answer 33

when refolding fails Hsc 70 chaperone can target misfolded proteins for ubiquitylation and degradation

Question 34

in SCF how is the 50 angstrom gap between the substrate bound to the f box protein and the ubiquitin on the e2 bridged

Answer 34

(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

Question 35

what are ubiquitin proteases

Answer 35

ubiquitin c terminal hydrolases that process ubiquitin precursor proteins to generate ubiquitn
approx 100 deubiquitylating enzymes which hydrolyse conjugated ubiquitin to regernerate monomeric ubiquitin

Question 36

how does the ubiquitin-proteasome system work

Answer 36

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

Question 37

what is the proteosome

Answer 37

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

Question 38

cervical cancer caused by human papilloma virus and ubiquitin systems

Answer 38

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

Question 39

maternally inherited deletion of UBE3A encoding E6-AP causes angelman syndrome

Answer 39

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

Question 40

ubiquitin system failure causing cancers

Answer 40

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

Question 41

ubiquitin system failure causing diseases involving membrane proteins

Answer 41

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

Question 42

chronic neurodegenerative disease from ubiquitin system failure

Answer 42

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

Question 43

other ubiquitin like proteins

Answer 43

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

Question 44

example of a ubiquitin like protein (UBL)

Answer 44

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

Question 45

sumoylation can compete with other lys modifiers

Answer 45

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

Question 46

regulation of sumo by reactive O2 species

Answer 46

oxidise reactive rhiols on sumo enzymes
Uba1/Ass1-s-s-Ubc9
sumo can not attach to proteins if they are not sumoylated

Question 47

examples of sumo functions (RanGAP1, IkB, c-JUn, p53 and mdm2)

Answer 47

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