Ubiquitination Flashcards
Post-translational modifications: what is it, examples
-Can be the addition of chemical group
Phosphorylation: phospho group (small negatively charged) revesibly conjugates to substrate, changing the chemical environment of residue it’s added to to ex. turn enzymes on/off). Kinases use ATP as a phosphoryl donor to phosphorylation proteins
Ubiquitination: polypeptide is reversibly added to sugars, lipids, etc (compared to phosphorylation, large)
Proteolysis:
Irreversible cleavage of protiens
Acetylation:
Reversible addtion of acetyl group to a protein (on 85% of proteins N-term.)
-Changes protein behaviour after it’s synthesised and functioning)
Ubiquitin: ubiquitination example, associated diseases
E1 (single point mutation in conserved exon 15 of UBE1) – X-linked infantile spinal muscular atrophy: congenital disorder involving severe contractures, scoliosis, chest deformities and death from respiratory insufficiency within months of birth due to progressive loss of anterior horn cells in the spine (neurodegeneration).
Specific effect of mutation on E1 activity is unknown but one reported mutation lowers overall expression levels
E2 – several cancers, mental retardation syndromes, Fanconi anaemia
E3s – Parkinsonism, hypertension, AML, Angelman syndrome, Fanconi Anaemia, etc
Parkinson’s:
Common disease usually of old age (120,000 cases in UK)
Neurodegenerative disease primary on neurons in substantia nigra (controls movement and coordination through dopamine production).
As PD progressive, dopamine levels in brain decreases and movement control diminishes. Initial symptoms are mostly motor based:
Tremor (hands, arm legs)
Bradykinesias (slowness of movement)
Rigidity (stiffness of limbs and trunk)
Postural instability (impaired balance/coordination
Other brain regions can also be affected (olfactory dysfunction, depression, dementia)
There is currently no disease-modifying agents for PD and current treatment focuses on symptom control (dopamine replacement therapy)
DUBs – spinocerebellar ataxia, neurodegenerative disorders, several cancers, immune disorders
Ex. Proliferating cell nuclear antigen (PCNA, few ubiquitinated structures) is important in DNA repair.
K164 is ubiquitinated as a signal to recruit polymerases to bypass DNA damage allows continued replication beyond that site
Fanconi anemia
19 independent gene mutations have been identified to cause FA (ex. E2 UBE2T and E3 FANCL).
Occurs in 1 in 100,000 live births and carrier frequency of 1 in 300.
Autosomal recessive disorder first described by Guido Fanconi in 1927. FA patients are often short with bone marrow failure (anaemia, cure with bone marrow transplant, hard to differentiate FA anaemia with others until chemotherapies are given and condition), developmental abnormalities, organ defect and an increased risk of squamous cell carcinomas and head and neck tumours
Patients with FA and BRCA mutations develop leukaemia at median age of 2, other FA patients at 13.5 years. Few survive beyond age 20
Primary defect is in repair of DNA damage, specifically of interstrand crosslinks (occurs during S phase.
FANCL binds Ube2T to monoubiquitinate FANCD2 (signals downstream repair factors including 3 in the BRCA pathway; FANCD1/BRCA2, PALB2, and BRIP1) and homologous substrate FANCD1).
Loss/mutation of any of the 19 in the core complex leads to reduction of FANCD2 ubiquitination, resulting in abnormal chromosomes and FA
Interstrand crosslink repair is regulated by cycle of both mono and deubiquitination
FANCD2 loss is used as diagnostic for FA (K561 is a key signal )
Removing DUBs that targets this complex, disrupts repair: USP1 (catalytic triad that’s upregulated in several cancers) and UAF1 (stimulates activity of several USPs)
Ex. ML323 inhibits USP1 via displacing and replacing beta strands, breaking hydrophobic core and taking place of residues. Also induces conformational change in catalytic site
PINK/Parkin model of PD (how it leads to disease, how to cure, )
Mutations (80 single aa substitutions including 12 in linkers) in parkin (RBR, autoinhibits via N-terminal Ub like domain) lead to autosomal recessive juvenile parkinsonism
Parkin is encoded by gene PARK2 and found in 2000 to have ubiquitin ligase activity and has ~450 substrates. Toxic substrate hypothesis made that those with mutant have build up toxic substrate (parkin substrate still unknown)
Parkin promotes (not necessary for) mitophagy (autophagic degradation of damaged mitochondria)
Parkin is activated via phosphorylation of Ser65 in Ubl domain and ubiquitination, both done by PINK1 (mutations also lead to Parkinson’s)
PD is likely caused by the accumulation of non-functional mitochondria in neuronal cells.
PINK1 (PARK6) is a kinase. Phosphorylates Ub which binds parkin. The UBL of parkin rearranges (WT parkin is inactive) and can be phosphorylated by PINK. Parkin then ubiquitinates everything in mitochondria, leading to mitophagy.
To reinstate mitophagy, can use:
PINK activators
PINK Phosphatase inhibitors
Parkin activators
USP30 inhibitors
Ubiquitin: binds other proteins and diseases associated
Attaches to protein’s lysine via isopeptide bond (amino group of lysine side group with C-term oxygen attached).
E1 (activating enzyme): 2. ATP dependent. Has ATP binding pocket.
E2 (): ~40 and binds cysteine. Associates with E3
E3: ~600 (30 HECT, 12 RBR, ~600 RING) so many diseases associated with E3. confers substrate specificity and transfers ubiquitin from E2 (usually).
Substrates: 10,000s
DUBs: ~100. Reversible (protein deubiqtuitinase that hydrolyses isopeptide bond).
Ex. cell cycle is regulated by ubiquitination (changes in abundance at different points in cell cycle is due to changes in gene expression and degradation via ubiquitin). When dyregulated, neurodegenerative diseases (alzheimers, parkinsons, etc) there are ubiquitin aggregates so may have some role in protein stability
Very important process that controls anything requiring signalling and many important cell processes, so leads to significant effects when mutated
Ubiquitin structure and it’s ubiquitination
Globular protein (8kDa) that when added completely changes proteins chemical and physical face and so how other proteins may interact with it.
At least 16 Ub like proteins (UBLs) in cell. Thought to come from bacterial ancestry in which they are used to make cofactors like thiamine. Each system has it’s own machinery that follows the same pattern
Can be ubiquitinated and position dictates function
Mono-UB: Endocytosis
K6: DNA damage response
K11: Cell cycle control
K27: Nuclear translocation
K29: WNT signalling
K33: Golgi trafficking
K48: Proteasomal Degradation
K63: DNA damage response /
Innate Immunity
M1 (methionine): Innate Immunity
Position of the next Ub also dictates function since overall structure of di-Ub is very different depending on the K it’s placed on. Structural biology allows us to see difference in where Ub can be added on the protein
Process of Ub activation and interaction with Ub
Ubiquitin is synthesised as precursor in which multiple are fused to one another on the N terminus of ribosomal proteins L40 and S27a
It is processed by UCH-type cysteine proteases to mature form that cleaves to expose Ubs C-terminal di-glycine motif important for substrate conjugation
Mature Ub is adenylated/AMPylated by E1 under ATP hydrolysis: E1 (open conformation) hydrolysis ATP to AMP.
The AMP binds Ub (adenylated state) via forming a phosphodiester bond between the hydroxyl group of the C-terminal GG motif and the AMP phosphate group (closed conformation)
Step 2: Ub is transferred to a catalytic Cys and AMP is released. Results in a thioester linkage between the C. terminal carboxyl group of Ub (GG) and the E1 cysteine sulfhydryl group (thiolated state).
Another Ub diffuses into the E1 and is adenylated (doubly loaded state).
E1 is only controlled by rate of diffusion so, constantly cycles Ub (as one gets discharged they other gets diffused up).
Differences/Similarities between ubiquitination and phosphorylation
Phosphorylation:
-Small negative charge
-One step of kianse transfering phosphoryl group from ATP to substrate
Ubiquitination:
-Larger protein
-Step-wise series of enzyme reactions
-Stable covalent modification
Both:
-Reversible
C-term. of protein (where ubitin attaches) is usually completely inert (else every protein will be fused to all others)
E2 mechanism and interactions
Kinases are good drug targets (kinase inhibitors) since has similar morphology of ATP binding pocket
E2 is a small globular enzyme. When E1 is in the doubly loaded state (binds this state most favourably), E2 causes transthiolation reaction (moving Ub from E1 Cys to E2 Cys). This results in the E1 being in the Adenylated state with one Ub
Ub charged E2 enzymes can work with 3 types of E3 enzymes: Homologous to E6-AP Carboxyl Terminus (HECT), RBR, or RING E3 ubiquitin ligases
E3 ubiquitin ligases
Ub charged E2 enzyme can bind 3 types of E3 enzymes:
-Homologous to E6-AP Carboxyl Terminus (HECT). Comprised of C-lobe (with catalytic Cys), N-lobe then substrate binding domain
E6-AP/UBE3A is HPV associated protein that ubiquitinates p53 infected cells contributing to the virus oncogenicity
Transthiolation from E2 to E3 catalytic Cys in C-lobe. HECT then transfers in another transthiolation to substrate. E3 substrate binding domain binds substrate and can form an isopeptide bond between the Ub C-terminal glycine and the amino group of an internal Lys on the substrate transfer Ub from C-lobe to Lys substrate
-RBR (RING-Between-RING)
Comprised of RING1, In-Between-RING (IBR), RING2 (not actually a RING protein. Doesn’t form cross-brace ring finger structure, forms linear binding fold instead and doesn’t have catalytic Cys so not an E2 recruitment module), then substrate binding domain.
E2 binds RING1, then hypothesised it transfers Ub to RING2 Cys, then from RING2 to substrate
-RING (Really Interesting New Gene)
Has unique globular cross branch domain that coordinates 2 Zn2+ molecules through specifically spaced Cys and His residues
Does not contain an active site Cys nor does it form a covalent link with Ub.
Acts as scaffold (not an enzyme) for E2 and substrate
RING domain binds Ub charged E2 and the substrate binds the substrate binding domain. Ub is transferred directly from E2 to substrate without the formation of a Ub-E3 intermediate.
Can’t mutate active site to infer function since not an enzyme, so instead mutate residues in Zn binding motif. However this does not result in point mutation that disrupts activity, actually prevents protein folding
DUBs
Energy intensive to add Ub
Reasons to reverse:
Change localisation
Easier for protein to enter proteasome for degradation
Reuse Ub elsewhere
Reasons for protein degradation:
Allows temporal control of systems
Response to oxidative stress, error in chaperones, etc causing protein misfolding.
May be cysteine proteases (79) or Zn coordinating metallo-proteases (12)
Steady state protein ubiquitination level is result of the rate of Ubiquitination compared to rate of DUBs
Ubiquitin-proteasome system
UPS discovery won Nobel prize in 2004
Major system for targeted protein degradation in cell which serves a regulatory function and important in protein quality control
Has regulatory and housekeeping functions.
Has proteins that ensure Ub is recycled
26S proteasome recognises K48 linked poly-ubiquitinated proteins and degrades
Proteasome is a large molecular machine
2000 kDa of 70 subunits
Middle is the proteolytic machinery the top is the substrate recognition and regulation
1) Recognition: Subunit Rpn10 of the proteasome regulatory particle recognizes the quaternary structure of the polyubiquitin chain on the substrate.
2) Binding: Subunits Rpn1 and Rpn2 of the proteasome regulatory particle bind the substrate. Uses ATP
3) Unfolding/Channel gating: ATPase subunits of the proteasome regulatory subunit unfold the substrate.
The interaction of ATPases Rpt2 and Rpt5 with α-subunits of the core proteasome causes the gating of the channel.
4) Subunit Rpn11 of the regulatory particle and DUBs Uch37 and Usp14/Ubp6 detach Ub from the substrate.
5) Translocation/Proteolysis: Translocation of the substrate polypeptide chain into the proteolytic chamber of the proteasome is facilitated by subunit ATPases of the regulatory particle, and hydrolysis of the peptide bonds releasing peptides (3-25 aa) is accomplished by protease subunits β1, β2, and β5
How do you prove that a protein is ubiquitinated?
- Is the protein modified in cells (molecular mass shift)?
- Does the shift in molecular mass correspond to the size of UB (8kDa)?
- Can I remove the modification with DUBs – in cells or in vitro
- Can I purify the modified protein and detect ubiquitination by mass spectrometry
- Can I in vitro reconstitute ubiquitination (need to know the E3)?
Add ubiquitin, E1, E2, E3, substrate, Mg2+, and ATP. Can add TCEP which is a reducing agent to prevent oxidation of catalytic cysteine and reduce thioester bond to aid proper ubiquitination.
Analyse reaction with coomassie staining
Can see band of intermediates: E1-adenylate form, E3-Ub, E2-Ub and Ub.
Mutate substrate’s catalytic Cys and identify if it’s involved
Drug targets in Ubiquitination process and examples of a drug (3)
E1
E2
HECT E3
RING E3
HECT/RING-substrate complex
E2-E3 complex
DUB
Proteolysis targeting chimaeras (PROTACs) are being developed to target disease causing proteins for degradation.
If successful can target non-druggable proteins, a breakthrough for many diseases
Proteasome:
In 1993 Myogenics was founded to identify a compound that attenuates (doesn’t completely inhibit) proteasome function in muscle to slow muscle atrophy.
Initial strong doubts in feasibility inhibiting proteasome due to many essential roles of UPS in critical biological processes
Velcade: First UPS drug to be approved. Inhibits proteolytic activity of proteasome by binding the catalytic site.
Treats multiple myeloma (produces a lot of unfolded proteins) very effectively (from certain death to chronic condition) since the build up of unfolded proteins overwhelms the cell leading to cell death.
Cancerous cells are more susceptible to drug
Thalidomide (immunomodulatory drug; IMID): Sold to pregnant women to treat morning sickness but caused severe developmental defects and surviving children had sever deformations of the limbs. Mechanism is unknown
Drug has 2 mirror image forms: (R)-enantiomer has sedative effects whereas (S)-isomer (10 fold stronger binding to CBRN and inhibition of self-ubiquitination) is teratogenic. Under biological conditions the isomers interconvert so separating the isomers before use is ineffective.
Drug is now used to treat multiple myeloma and leprosy. Recently found thalidomide’s target is cereblon.
Thalidomide binds cereblon promoting protein protein interactions through one part binding cereblon and the other binding the substrate, acting like a molecular glue (similar to mechanism of PROTACs).
Instead cereblon acts on other substrates it doesn’t typically and the protein degraded is necessary to prevent teratogenicity
Cereblon when active forms an E3 ubiquitin ligase complex with DNA binding protein 1 (DDB1), RBX1 a RING box protein and Cullin 4A/B (Cul4A/B) important for limb outgrowth
As well as it inhibiting E3s towards it’s normal substrates and have anti-cancer effects via promotion of CRBN mediated degradation of B-cell specific Ikaros type transcription factors IKZF1/3
