Lecture 13 ubiquitin code Flashcards
What does mono ubiquitination lead to?
Trafficking, endocytosis, gene expression, protein interaction.
What does K6, K11, and K29 ubiquitination lead to?
Proteasomal degradation and DNA repair.
What does K33 ubiquitination lead to?
Protein interaction
What does K48 ubiquitination lead to?
Proteosome degradation
What does K63 ubiquitination lead to?
Kinase activation, DNA repair, trafficking, translation
What are the functions of linear chains and forked chains?
Linear= signalling pathway activation. Forked= Unknown.
What is the confirmation of lysine 63 compared to lysine 48?
63 is an open extended confirmation where as 48 has multiple confirmations. Chemically identical but structurally different. The different 3 dimensional topology is read by ubiquitin receptors.
Compare K63 di-Ub to linear di-Ub.
63- Glycine 76 at top of ubiquitin and lysine 63 at bottom. Linear has methionine linked to glycine 76. Similar in tertiary structure to 63.
What is the lowest energy structure for K48?
The close confirmation where it is interaction non covalently and covalently through isopeptide bond. Has an equilibrium.
What is the lowest energy state of K63?
Does not have the same equilibrium. Beads on a string concept. Open confirmation.
How do ubiquitin receptors bind?
When ubiquitin builds up as a substrate modification then it gets recruited by a receptor which relays signals through other protein protein interactions.
What is the ubiquitin code?
Structurally unique (poly)ubiquitin linkage types recruit distinct sets of effector proteins (ubiquitin receptors) to influence the fate of modified proteins.
What are examples of re modelling ubiquitin chains?
E1/E2/E3s, Ub-R, DUB ( deubiquitinating) some E3s can also do this.
How can ubiquitin act as a scaffold?
Protein X has no interactions and gets poly ubiquitinated this allows protein A a ubiquitin receptor which binds non covalently. This is an intermediate. Can have direct and indirect interactions. Indirect: Protein X has ub receptor protein Z (attached to protein Y) bind to it to mediate an interaction between protein x and protein Y.
What are examples of proteins in the proteosome that are ubiquitin receptors?
- 65a subunit of 26S binds directly to lysine48 ubiquitinated proteins. 2. hHR23A bridge protein ub-R. Has a binding site for S2 proteosome subunit. This is the bridge between the substrate and the proteosome.
What is TRAF6 for?
A scaffold adaptive protein thats downstream of lots of different receptors, particularly in cytokine signalling where TRAF6 is activated in response to signal transduction. Modified with the polyubiquitin chain. A lysine 63 linked and recognised by Ub-R Tab2. TAB2 forms a stable complex with a kinase called TAK1. When TAB2 the receptor sees lysine63 linked polyub it binds avidly with noncovalent interactions. Brings TAK1 into the same complex and in this case TAB2 binding to lysine63 linked polyub changes TAB2 confirmation changing the confirmation of TAK1 and activates it as a kinase to phosphorylate other targets to produce a signal.
What affinity does ubiquitin bind with?
Very low affinity.
How are higher affinity reactions mediated?
Through avidity. By having multiple UBDs in the same Ub-R or oligomerisation (noncovalent interactions of ubiquitins on different chains more than one ubiquitin binding domain to more than one ubiquitin) of Ub-Rs. Avidity effect=multivalent interactions.
What reaction surfaces can ubiquitin reactions recognise?
Hydrophobic surfaces, acidic surface, C-terminal tail and others (see moodle snapshot). These are differentially exposed and positioned in polyubiquitin chains of different topologies.
What is avidity?
The accumulated strength of multiple affinities of individual non-covalent interactions. avidity levers weak interactions into higher affinity interactions.
What is an example of DUB with multiple ubiquitin binding domains for increased affinity in its primary sequence?
DUB USP5: ZnF UBP, UBP, UBA1, UBA2, UBP.
How does USP5 function?
Unanchored polyub chains act as second messengers. USP5 keeps those polyub chains at low levels so needs a high binding affinity for its substrate on polyub. Does this through three ub domains. Simultaneously recognises three ubiquitin in at least a tetra ubiquitin chain.
Where does the cell avidity come from?
The third domain called the ZnF-UBP domain recognises free C-terminal when not attached to a substrate protein. The unanchored polyub has a free glycine 76 tail for binding.
How does equilibria explain avidity increase?
The interaction of the first ubiquitin to the ubiquitin binding domain is weak and still in equilibrium. When a second ubiquitin is tethered to the first its brough into closer proximity to the second binding site. The effective local concentration of the second ubiquitin is much higher than the real concentration. The Kd increases (the effective binding constant). Cooperative binding where if either ubiquitin interaction to the receptor fails it will still be in close proximity.
What is selectivity?
Binding of UBDs to the right polyubiquitin chain.
What is an example of selectivity?
The closed confirmation of K48-linked polyubiquitin chains (plus K6, and K11) arises as two I44 surfaces on neighbouring ubiquitins participate in a non covalent (hydrophobic) interaction. This affects what UBD interaction surfaces are presented in the chain.
What is an example of UBDs selectively recognising specific isopeptide linkages in a polyubiquitin chain?
UBA domain of hHR23A binding to K48-linked polyubiquitin. Avidity: Simultaneously interacts with two I44 surfaces on adjacent polyubiquitins. Does this by wedging into the cleft between the closed and open confirmation of lysine48 linked polyub dimer. The UBA domain has two hydrophobic surfaces and both simultaneously interact with the isolation psi44 on one ubiquitin and the other. Two weak interactions to form a strong interaction. Selectivity: Only lysine48 linked polyub positions itself properly to match the hydrophobic sites on the UBA domain. No other dimer is properly positioned in this way.
How do tandem multiple UIMs (ubiquitin interaction motifs) affect reading the ubiquitin code?
Multiple UBs are optimally positioned to correspond to the confirmations that are unique to the polyub chains and these chains are constantly moving but they will have the lowest energy structures.
What experiment showed the importance of the poly ub specificity?
Rab80 has UIM1 and UIM2 modifications. Introduced different mutations to the linker length. Kd massively increased from 3.6 to 85 in one example.
How do ubiquitin receptors work in trafficking?
Ub-R1 and Ub-R2 both recognise the same interaction surface on Ub. Ub-binding affinity of Ub-R2 is higher than Ub-R1. Trafficking occurs as individual UBD-Ub interactions are weak and competition for binding means Ub-R(1) will hand off cargo to Ub-R2.
