Extra

Question 1

How cAMP signaling activated, what is cAMP, effector proteins, desensitisation

Answer 1

Cyclic AMP is a small molecule that serves as an intracellular second messenger of many hormones & neurotransmitters

Generate inside cells by the action of plasma membrane-adenylyl cyclase and quickly diffuses throughout cell (even through nucleus)

cAMP is detected by PKA, EPAC, CNGCs, POPEYE domain proteins (POPDC)

PDE is only family that degrades cAMP into AMP. Different localised degradation methods depending on type and the subcellular localisation is integral to function

Question 2

Targets of the cAMP pathway for clinical applications and examples

Answer 2

Wide variety of receptors that are able to regulate adenylyl cyclase activity (ex. GPCRs)

Multiple adenylyl cyclase isoforms (9/10) - Forskolin activates and researched for use in heart failure

Cell type specific expression of targets: PKA, EPAC, CNGC, POPEYE

PKA: phosphorylates 1500 proteins, inhibitor drugs have detrimental effects as not specific enough

PDEs

Question 3

PKA: substrates, isoforms - difference, concensus sequence

Answer 3

PKA phosphorylates 1500 prot2 cAMP binds to regulatory subunit, disassociating complex, releasing catalytic subunits to phosphorylate target substrates

2 isoforms of regulatory subunit: PKA-RI (predominantly cytosolic to monitor gradients of cAMP in cells; weak or no AKAP binding) and PKA-RII (anchored at specific intracellular sites (organelles, membranes, etc) by strong binding to AKAPs)

Role of PKA catalytic subunit isoforms is unknown

Consensus sequence: R-R-X-S/T

Question 4

AKAPs function, mechanism, use in clinic

Answer 4

A-kinase anchor proteins that sample cAMP gradients in cells. There are AKAPs for every major cell location (nucleus, FA, membrane, etc) to bind PKA to specific locations.
Some AKAPs can interact with other proteins such as PDEs, kinases, etc for faster spatiotemporal control of signalling

PKA-RII has a two alpha helix interface that the AKAP peptide binds with high affinity.
As a PKA inhibitor, can produce pseudo-substrate with the PKA consensus motif that AKAP will bind instead, causing improper localisation of PKA - non clinically approved yet

Question 5

EPAC: isoforms, difference, function, regulation, clinic

Answer 5

EPAC has two isoforms (Epac-1; wildly expressed and Epac-2;), with Epac-2 having 2 cAMP binding domains
EPAC is autoinhibited at rest by the GEF domain blocked by the regulatory domain aand binds cAMP to activate (with a lower affinity than PKA)
EPAC is a GTP exchange factor (GEF) to mini-G-proteins Rap1 (stabilises VE-cadherin mediated cell-cell contacts, activates ERK, and induce SOCS-3) and Rap2

No small molecules that activate or inhibit EPAC successfully

Question 6

Use of cAMP pathways as a vasorelaxant

Answer 6

PDEs: Caffeine is a mild bronchodilator that inhibits PDEs (especially PDE4), causing increase in cAMP, activating PKA, causing smooth muscle relaxation and bronchodilation (opening airways)

GPCR: Salbutamol activates beta-2 receptor for bronchodilation in asthma
Activates AC producing cAMP that activates PKA (also activating EPAC).

EPAC: Activates Rap1 small GTPase that leads to activation of ryanodine receptor in vascular smooth muscle cells, releasing localised Ca2+ sparks.
These sparks activate K+ channels, inducing membrane hyperpolarisation, which inhibits voltage gated Ca channels (VGCC) reducing Ca influx.
EPAC also activates Na+/Ca2+ exchangers (NCX) that pumps Ca out cell

Ca2+ is required for smooth muscle contration and its removal prevents actin-myosin interaction - promoting vasorelaxation

Question 7

POPEYE: discovery process, function, mechanism, structure, associated diseases

Answer 7

Incubated cell lysate with cAMP coated magnetic beads, seperacted with magnetic cell sorting and did proteomics (MS) to identify proteins bound
Discovered protein POPDC with a canonical cAMP binding site. Has 3 membrane binding domains

Theory of how it functions: POPDC binds ion channels to activate. Binding cAMP removes it from ion channels stopping activation.

High expressed in cardiomyocytes and involved in pacemaking. Mutation causes cardiac arrhythmia and muscular dystrophy in patients

Question 8

PDEology: types, how they arise, localisation, differences, similarities

Answer 8

Cyclic nucleotides require large diversity of PDEs
11 different families and >100 different isoforms from these families arising from being encoded by multiple genes and alternative mRNA splicing

Often localisation is directed by the N-terminal targeting domain (highly vaired in PDE4 isoforms) allowing a single isoform to have multiple non-redundant roles in different tissues, cells and microdomains within the same cell

Differ in:
Ability to hydrolyse cAMP and cGMP (Vmax and Km values)
Regulatory properties
Intracellular localisation
Signalling complexes recruited to

Active sites (catalytic region of 360 aa) are almost identical so need a way to target PDE targeting drugs for use in clinic (ex. viagra is a PDE5 encoded by only one gene so easy to target without many off-targets)

Question 9

PDE activation effect, clinical examples, mode of action for inhibitors and activators

Answer 9

Short term: PKA phosphorylates and activates PDE3, 4 and 8, reducing cAMP

Long term: PKA phosphorylates CREB inducing upregulation of PDE4 expression

PDE inhibitors (roflumilast; COPD and apremilast; psoriasis) induce the long term pathway, massively upregulating PDEs and making the drug less effective

PDE4 in clinic:

PDE4 inhibitors as antidepressants, cognitive enhancers, anti-fibrotic agents, anti-cancer and anti-inflammatory.

Ex. PDE4 is highly expressed in immune cells. Inhibitors decrease proinflammatory cytokine production (ex. TNFalpha) and increase PKA activation of CREB which increases transcription of IL-10 cytokine.

Stimulate PDE4 (small molecule binding PKA consensus sequence) in polycystic kidney disease (reduce cyst growth promoted by cAMP) and prostate cancer

Question 10

PDE4: isoforms, how they arise, general structure

Answer 10

4 isoforms - PDE4A-D, and each gene produces multiple isoforms via alternative splicing and alternative promoter usage

Isoform groups:
Long (UCR1 and UCR2)
Short (UCR2)
Super-short (part of UCR2)
Dead-short (catalytically inactive)
Diversity in activation mechanism
Each has a conserved catalytic subunit (differs enough for diversity in targeting)

Regulatory domains (UCRs):
UCR1 - PKA (R-R-X-S/T-X)
Some PDE4As don’t have ERK site and 4A long forms can’t be SUMOylated
UCR2 - Forms autoinhibitory domain that interacts with catalytic site (especially in long-forms)

C-terminal region: sub-family specific (same in all PDE4Bs) and typically involved in substrate interactions (governs protein-protein interactions (ex. interaction with AKAPs)), target for Ab, etc
ERK (P-X-S/T-X) phosphorylation site in C terminus. Near ERK consensus site is a MAPK docking domain to allow ERK binding

N terminal region: Isoform specific (different in long/short/etc) specific postcode sequence to target the PDE to cell compartment

Question 11

Activation of each PDE4 isoform

Answer 11

Long form:
Low basal activity due to autoinhibition

Autoinhibition: Exists as dimer in which UCR1 and UCR2 tansmolecularly interact to form regulatory module and transcaps the other, allowing UCR1 to block the catalytic site where cAMP binds

PKA phosphorylates PKA consensus site in all sub families long forms (R-R-E-S-Xphobic in UCR1), causing PDE4 activation (opens up the transcap by weakening UCR1 and UCR2 interaction / allows disassociation of regulatory domain), increasing Vmax of cAMP hydrolysis

SUMOylation: small protein added to Xphobic-K-X-E with PKA phosphorylation locks in an open conformation and protects against ERK phosphorylation

ERK phosphorylation in the UCR2 region transiently decreases activity - enhances autoinhibition, decreases PDE4 long form activity, cAMP increases, PKA phosphorylates PKA site on long form to increase activity
Found by adding ERK activator and observed a small inhibition of 25% (for cAMP signalling small differences is important) in long and super-short

ERK phosphorylation in catalytic domain promotes conformational change in which C-terminal helix forms an intramolecular cis cap to prevent activity and block the catalytic domain

Scaffold proteins (ex. RACK1, beta-arrestin) can bind the core catalytic unit to alter gating by UCR2 and the C terminal helix.

Short form:
No response to PKA activation (don’t have UCR1 to be phosphorylated)
ERK phosphorylation increases activity
Monomer
More active than long form despite same catalytic domain since doesn’t have UCR1 and UCR2 transcap in dimerisation

Super short form:
ERK phosphorylation decreases activity
No response to PKA activation

Dead-short form: catalytically inactive due to severe N and C terminal truncation

Question 12

PDE4 inhibitors: disease treated, signalling pathway, side effects, binding

Answer 12

Roflumilast to treat chronic inflammation in COPD. Very bad side effects so only use in very severe cases
In COPD, cAMP drives anti-inflammatory responses so aim to increase levels with PDE4 inhibition
Increase of PKA activation phosphorylates Csk which inhibits LPS (inducer of inflammatory responses; TNF-alpha, ILs)
T cells treated with LPS has large increase in PDE4 (especially the short form) to inactivate
Nausea and vomiting (associated with PDE4D inhibition and CNS penetration), diarrhea, etc
High affinity rolipram binding site; HARBS binding is associated with nausea, vomiting and headaches from BBB penetration of small molecule. HARBS is PDE4 long forms, and PDE4D is highly expressed in brain so is the main contributor of CNS symptoms (via HARBS binding)

Due to mode of action and lack of sensitivity for isoforms/subfamilies, since they are important in inflammatory and non-inflammatory functions

Apremilast is highly selective for PDE4s but no selectivity within the family.
Drug to treat psoriasis- can cause complete remission (highly successful)
Less side affects since poor brain transmission and low binding to HARBS
However still causes, nausea, respiratory tract infection, etc
Better therapeutic index but higher dose = more severe symptoms

Both are competitive with cAMP

Question 13

PROTAC: mechanism, example, advantages, disadvantages

Answer 13

Drug that doesn’t depend on active site inhibition
Recruits ubiquitin to POI to degrade- takes longer to work than direct inhibitor binding

Fused 4B specific compound to linker and E3 ligase.
KTX207 is extremely successful degrading shortform PDEs (dimerisation stops linker from recruiting E3 ligase)

Ex. E3 ligase cereblon targeted to with Pomalidomide ligand to degrade PDE4B

Advantages:
Selectivity (short vs longforms) via warhead and linker design
Degrades protein ablating enzymatic and non-enzymatic functions (ex. some PDEs may act as scaffolds)
Enzymatic mechanism works at low doses - acts sub-stoichiometrically with one PROTAC degrading multiple of the protein molecule (less side effects)
Very long lasting as once inside cell PROTAC keeps working and is not consumed - longer pharmacodynamic window
No rescue from upregulation of target protein

Disadvantages:
Low bioavailability - poor permeability from high MW
Low BBB penetration for CNS - high MW
Has to recruit E3 ligase in cell and if not well expressed PROTAC will not work
Hook effect- concentration has to be in ‘Goldilocks’ window - to high and binary binding of POI or E3 can dominate (PROTAC binds one or the other)

Question 14

Ideal PDE4 inhibitior

Answer 14

High affinity for family, PDE4 specific
Isoform specific (ex. PDE4B2) and if not, subfamily specific (PDE4Bs)
If not, non-brain penetrating to decrease CNS side effects ex. nausea
Low HARBS binding - minimise nausea, linked to PDE4D mainly
Moderate LARBS binding - anti-inflammatory action in peripheral immune cells, however LARBS binding also causes increased gastric acid production - gastrointestinal issues, etc
No effect on other active proteins (ex. kinases)
Good bioavailability - ideally oral with min. 8-12 hour t1/2 for once daily dose, moderate clearance, minimal active metabolites, etc

Question 15

Associated diseases with Ubiquitination protein mutants, example of Ub in normal cell and function

Answer 15

E1 (single point mutation in conserved exon 15 of UBE1) – X-linked infantile spinal muscular atrophy:

E2 – several cancers, mental retardation syndromes, Fanconi anaemia

E3s – Parkinsonism, hypertension, AML, Angelman syndrome, Fanconi Anaemia, etc

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

Ex. Tumour suppresor p53 is downregulated in many cancers
E3 ligase MDM2 targets p53 for degradation, and is often targeted in treatment to inhibit, causing enhanced cell cycle arrest and apoptosis in tumour cells

Question 16

X linked infantile spinal muscular atrophy: symptoms, cause

Answer 16

Single point mutation in conserved exon 15 of UBE1
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 and causes build up of damaged proteins causing loss of neurons

Question 17

Parkinson’s: prevalence, cause, symptoms

Answer 17

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)

Question 18

Fanconi anaeia: discoverer, cause, prevalence, symptoms, signalling, regulation

Answer 18

Mutations in 19 independent genes 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) , developmental abnormalities, organ defect and an increased risk of squamous cell carcinomas and head and neck tumours
Hard to differentiate FA anaemia with other aplastic anamias until chemotherapies are given

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 and FANCI which signals downstream repair factors including 3 in the BRCA pathway; FANCD1/BRCA2, PALB2, and BRIP1
After repair, FANCD2 and FANCI are deubiquitinated by USP1-UAF1 complex to turn off pathway
Loss/mutation of any of the FA core complex leads to reduction of FANCD2/FANCI 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
Impairs DNA repair and potentially causes cancer cells to be sensitive to DNA-damaging treatments

Question 19

PINK/Parkin model of PD (cause, parkin structural features, signalling in normal and damaged cells)

Answer 19

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

Toxic substrate hypothesis - those with parkin and/or PINK1 mutant have build up toxic substrate (parkin substrate still unknown) typically cleared by mitophagy that is a key factor in the degeneration of dopaminergic neurons in PD

Normal:
PINK is translocated into mitochondria via TOM/TIM and then degraded
This relies on membrane potential - first thing dysregulated in mitochondrial damage

Mitochondrial damage:
PINK1 accumulates on the OMM where it phosphorylates Ub on mitochondrial proteins
The autoinhibited parkin in cytosol binds phospohrylated Ub and now at mitochondria, PINK1 can phosphorylate parkin at Ser65 in UBL domain
Induces conformational change that exposes key residues, activating parkin’s Ub ligase activity.
Parkin adds Ub to mitochondrial proteins (~450 substrates), signalling damaged mitochondria for degradation
Feedback loop allows PINK1 to phosphorylate to recruit more parkin
This promotes (not necessary for) recruitment of autophagic machinery to degrade damaged mitochondria through mitophagy

Question 20

Ubiquitination: chemistry of Ub attachment, proteins involved, number of each, example of process it’s important in

Answer 20

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 (conjugating enzyme): ~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).

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

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

Question 21

Ubiquitination: general principle of how it works to alter protein behavioiur, polyubiquitin chain functions

Answer 21

Globular protein (8kDa) that when added completely changes proteins chemical and physical face and so how other proteins may interact with it.

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

Question 22

Process of Ub activation and interaction with Ub

Answer 22

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

Question 23

E2 mechanism, interactions and why multiple types

Answer 23

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) binding to Ub via thioester bond. This results in the E1 being in the Adenylated state with one Ub

Different E2s:
Partner with distinct E3s
Preferentially build mono or a particulalry linked polyUb chains
Can be differentially expressed, or post-translationally modified to respond to particular signals

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

Question 24

E3 ligases: names, structure, how they add Ub

Answer 24

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) - RING-HECT hybrid mechanism
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 has 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

Question 25

DUBs: how, reason to do, reason to degrade protein

Answer 25

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

Question 26

Ubiquitin protease system: function, process

Answer 26

Major system for targeted protein degradation in cell which serves a regulatory function - important in protein quality control and housekeeping functions - degrades proteins no longer needed 26S proteasome recognises K48 linked poly-ubiquitinated proteins and degrades Proteasome is a large molecular machine 2000 kDa of 70 subunits 20s core particle is the proteolytic machinery and the 19s regulatory particle (RP) is responsible for substrate recognition and regulation 1) Recognition: Several proteins are involved in recognition, and RP subunits Rpn13, and Rpn10 directly binds the quaternary structure of the polyubiquitin chain on the substrate. Ensures only appropriately tagged proteins enter 2) Binding: RP subunits Rpn1 and Rpn2 bind the substrate (uses ATP) to help it interact with the proteasome 3) DUBs Rpn11 (RP), Uch37 and Usp14/Ubp6 detach Ub from the substrate. 3) Unfolding/Channel gating: RP ATPase subunits (ex. Rpt2 and Rpt 5) unfold the substrate (must be linear to be processed). The interaction of ATPases Rpt2 and Rpt5 with α-subunits of the core proteasome causes the gating of the channel. 5) Translocation/Proteolysis: Translocation of the substrate polypeptide chain into the proteolytic chamber of the proteasome is facilitated by subunit ATPases of the RP, and hydrolysis of the peptide bonds releasing peptides (3-25 aa) is accomplished by CP protease subunits β1, β2, and β5

Question 27

How to prove a protein is Ubiquitinated

Answer 27

-Is the protein modified in cells (molecular mass shift of 8kDa) -Can I remove the modification with DUBs in cells (overexpress DUB) or in vitro -Can I purify the modified protein and detect ubiquitination by mass spectrometry (obtain info on which K of Ub linkage) -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 involve

Question 28

How to reinstate mitophagy in PINK/parkin signalling and potential downside

Answer 28

PINK activators - increase stability/activity PINK Phosphatase inhibitors to enhance kinase activity Parkin activators - molecules found to activate parkin in the absence of PINK1 too USP30 inhibitors - removes Ub chains on mitochondrial proteins, inhibiting increases mitochondrial Ub and promotes parkin activity Excess or unregulated activation of mitophagy can cause overzealous mitochondrial turnover and lead to cell stress, or excessive cell death (especially in neurons and hear muscle cells)

Question 29

Main general strategies to treat T2D

Answer 29

Drugs must be in conjunction with change in diet and exercise, and requires regular monitoring, and ongoing care - eye exams, blood pressure, blood glucose and cholesterol monitoring, etc Need safer, and better drug treatments, though lots on the market -Reduce obesity -Improve pancreatic function/increase insulin secretion (FFA receptors. incretins, ghrelin, somatostatin, etc) GLP1 receptor agonist drugs (supresses appetite for weight loss; ozempic) TZD drugs (activates PPARgamma to improve insulin sensitivity and increase fat storage in adipose tissue lowering blood glucose levels) Islet cell transplant for severe cases, expensive -Improve skeletal muscle insulin sensitivity Exercise encourages glucose uptake by muscle cells lowering blood sugar TZDs Metformin improves insulin sensitivity and helps muscles better absorb glucose -Increase incretin release from the gut Drugs targeting GLP1 developed (ex. semaglutide agonist) and are safe and effective, was very difficult to develop -Reduce inflammation Association between increasing inflammation (particularly in adipose from infiltrating macrophages) with insulin resistance and onset of diabetes -Dietary change Reason why people become obese Fish oil omega 3 supplements to improve metabolic health Dietary fibre, it's fermentation in gut produces short chain fatty acids (anti-inflammatory effects and improve insulin sensitivity)

Question 30

Animal models for diabetes

Answer 30

Leptin deficient: LOF mutation in leptin or leptin receptor gene - causes insulin resistance and obesity Diet induced obesity: high fat diet - insulin resitance and obesity PPARgamma KO - insulin resistance and adipogenesis

Question 31

Fatty acids: types, length, receptors

Answer 31

Recommended dietary fat is 20-35% of energy intake (majority is LCFA) Saturated fatty acids (SFA) from animal fat and tropical oils Short chain fatty acids (SCFA, 2-6 C) from fermentation of fiber by gut microbiota Medium (MCFA, 7-12 C) Long (LCFA, 13-22 C) from fish, animal fat, vegetable oil Very long: >22 C Monounsaturated fatty acids (MUFA): Oleic palmitoleic Polyunsaturated fatty acids (PUFA): Omega-3, 6, 9 -Receptors Long chain FA: FFA1 (GPR40), FFA4 Short chain FA: FFA2, FFA3

Question 32

FFA1 and FFA4 expression profile and function for each

Answer 32

FFA1 expression profile: Mostly expressed in pancreas beta and alpha cells Enteroendocrine L, I and K cells (produce GLP1, CKK and GIP respectively) Skeletal muscle, Heart, Liver, Bone, Brain, Monocytes FFA4 expression profile: Lower intestine (enteroendocrine cells): L cells secreting GLP1 and I cells secreteing CKK Not pancreatic beta islets: low/negligible Pancreatic alpha: Increases glucagon secretion Pancreatic delta: Inhibits somatostatin secretion Adipose tissue: anti-inflammatory effect, insulin sensitisation (increases glucose uptake) Taste buds: fatty tase perception Intestinal epithelial cell: nutrient sensing Macrophages: anti-inflammatory Hypothalamus: decrease appetite and weight

Question 33

FFA1: Signalling pathway, ligands, ligand effects

Answer 33

FFA1 receptor/GPR40 Signalling: Activated by MCFAs, LCFAs Highest potency for long chain PUFA Can couple to Gq (primary pathway), i and Gs (PKA and EPAC enhance vesicle fusion with PM to increase incretin release) in a ligand dependent manner Little evidence it can recruit arrestin (desensitisation and internalisation of GPCRs to regulate cycling of FFA1) FFA1 contributes 50% of total effect of LCFAs on enhancing glucose stimulated insulin secretion (GSIS) Chronic effect inhibits GSIS and increase lipotoxicity (TAK-875 induced liver toxicity) However recent evidence shows FFA1 is protective of beta cell (reduces apoptosis or enhances function under stress) Previous evidence indicating lipotoxicity may be due to non-selective lipid toxicity due to chronic exposure of LCFA (regardless of receptor involvement) Agonists: Synthetic agonists: TAK-875 and AMG-837 (partial agonists), AMG-1638 (full agonist) which are all ago-allosteric modulators (amplify agonistic activity of endodgenous ligand alpha linolenic acid) All bind FFA1 to increase Gq signalling and calcium driven augmentation of insulin secretion in pancreatic beta cell AMG-1638 also increases insulin secretion and increases incretin release (GLP1 and GIP) through activation of Gs in full agonism (dual effect), so larger insulin secretion Synthetic antagonists also available

Question 34

Incretin: secreting cells, target, effect,

Answer 34

Gut derived hormones secreted from enteroendocrine L and K (produce GLP1and GIP respectively) into blood within mins of eating to regulate insulin secretion GLP1 Stomach: decreases gastric emptying Brain: increases satiety Macrophages, FFA4: anti-inflammatory GLP1 and GIP Pancreas, FFA1: augment insulin secretion and decrease glucagon secretion

Question 35

FFA1: current drugs on market, future of agonist development

Answer 35

Current drugs: TAK-875 reduced hyperglycemia and improved GSIS in type 2 diabetic patients Reached phase 3 clinical trials but found it induced liver toxicity Excessive FA in liver (steatosis) disrupts normal function (insulin resistance caused) and can exacerbate conditions like non-alcoholic fatty liver disease; NAFLD No current FFA1 drugs on the market as others withdrawn due to concerns on liver toxicity and safety profile long term Withdrawing the drug from clinical trials allowed research to be released for public access, so lots known Future: FFA1 potentially has multiple allosteric binding sites to target as therapeutic agent Understand how different FFA1 ligands potentially interact with each other to consider the effect of drug in the presence of high endogenous FA Unclear how natural ligands bind so further research to clarify the most effective way to target Understand mechanism behind hepatoxicity to produce safer drugs (drug chemical structure, not receptor activation, is causing toxicity since larger molecules are more toxic in metabolism)

Question 36

FFA4: activators, signalling, structure, variants, KO studies

Answer 36

FFA4 (GRP120) Activated by wide range of LCFA (particularly PUFAs) Effect of FFA4 is context dependent: In diet induced obesity mice models, FFA4 is upregulated Mixed data in FFA4 adipose expression reported in obese individuals (up or down regulation) High fed FFA4 KO mice developed insulin resistance (suggesting FFA4 protects against diet induced dysfunction) and omega-3 FA (ex. DHA a PUFA) failed to improve insulin resistance (improved in WT) However other studies show no significant effect of FFA4 KO on insulin sensitity in high fed mice, yet omega-3 improved insulin sensitivity (suggesting FFA4 is not essential for the metabolic effect) Studies vary in mice strains, housing conditions, microbiome, diet composition, compensatory pathways (others ex. receptors; FFA1, PPAR, etc may contribute to omega 3 effects) etc Gq - promotes GLP1 secretion Gi - inhibits ghrelin secretion Arrestin (key component) - anti-inflammatory effects Splice variants: Humans - short (most commonly expressed) and long (16aa insert in ICL3) forms of FFA4 Rodents - short form Short form - activate Gq and Arrestin mediated pathways Long form - preferentially recruits beta-Arrestin May have limited tissue expression (predominantly colon). Physiological relevance not fully understood

Question 37

FFA4: activation effect, KO effects

Answer 37

In pancreatic alpha-cells - increase glucagon secretion (elevates blood glucose) In pancreatic delta cells - inhibits release of somatostatin (inhibitor of glucagon and insulin release) Adipocytes: Activates Gq to increase glucose uptake and inhibit lipolysis - produces PDE3B and inihit the cAMP produced (inducer of lipolysis) Gastric ghrelin cells: inhibits release of ghrelin via Gi Ability of islets to secrete insulin unaffected by FFA4 KO However, FFA4 KO studies indicate a cytoprotective role for FFA4, aiding in inhibition of lipotoxicity Anti-inflammatory in macrophages: FFA4 activation by DHA recruits beta-arrestin which interacts with TAK1 binding protein (TAB1) preventing it from interacting with TAK1 (Transforming Growth Factor Beta Activated Kinase 1), blocking TAK1 activation (typically activated by LPS and TNFalpha) and suppressing its downstream inflammatory pathways (NF-κB and JNK) that would induce insulin resistance in adipocytes

Question 38

Ghrelin: function, expression profile, regulation, mode of action

Answer 38

Circulating appetite stimulant Produced predominantly in endocrine cells (alpha cells) of human gastric mucosa lining the stomach and proximal small intestine Rise in ghrelin before each meal (signal for meal initiation), decrease after eating Simulates GHSR to increase food uptake, body weight, insulin resistance, and decrease insulin release Inhibited by FFA2 and FFA4 (in ghrelin containing cells in the stomach, so is FFA3 at lower levels but role is less defined)

Question 39

FFA1 synthetic ligand binding, key residues, importance

Answer 39

Ligand binding: Earliest/most charecterised is TAK-875: carboxylate interacts with two Arg. Asparagine formed H bond with one of the Arg to position the residue within the binding pocket Allosteric binding protruding into the lipid environment of PM (between TM3 and TM4) - similar binding to analogous receptor S1P to the S1P1 receptor (between TMV7 and TM1) Key moment in FFA research was understanding how TAK-875 bound to improve efficacy, potency, and understand in general how FFA ligands bind (via PM) Another binding site between TM4 and TM5

Question 40

Adipocyte differentiation, regulation, experimental evidence

Answer 40

MSCs differentiated into preadipocytes and PPARgamma activation drives into adipocyte FFA4 has important role in progression - antagonist and siRNA KO mice reduces preadipocyte differentiation into adipocyte and inhibits PPARgamma expression Agonist in vitro causes slight increase Measured with Oil red O staining, and ex. RT-qPCR

Question 41

Characteristics of adipose tissue in diabetes

Answer 41

Macrophages increases their infiltrate adipose tissue as obesity progresses into chronic obesity (associate with diabetes) Switches from M2 (anti-inflammatory phenotype) into M1 (pro-inflammatory phenotype) Enlarged adipocytes from excess lipid storage

Question 42

Treating diabetes via FFA receptors: stimulate/inhibt, complications

Answer 42

FFA1: stimulate to enhance GSIS and potentially improve beta cell function and insulin sensitivity Cons: over stimulation could lead to beta cell exhaustion over time, so balance of alpha cell signalling to prevent excessive glucagon release, previous drugs led to liver toxicity FFA2: stimulate to promote gut-brain signalling to potentially improve insulin sensitivity and reduce inflammation, KO prevents beta cell mass expansion from insulin resistance Cons: May cause excessive inflammation (C2 increases ROS), disturbances in gut microbiota, SCFA are poorly absorbed, some studies show FFA2 (with C3) can inhibit GSIS, a FFA3: stimulate to promote glucose tolerance and insulin sensitivity Cons: may cause disturbance in gut microbiota, its role in glucagon secretion needs to be further investigated FFA4: stimulate to improve insulin sensitivity, reduce inflammation and enhance adipocyte function Cons: overactivation may cause excessive adipose tissue remodelling or lipotoxicity, effects are tissue specific so targeting is important

Question 43

Short chain FA: receptor, source, involvement in diabetes

Answer 43

SCFA receptor: FFA2 and FFA3 Modulation of gut bacteria (trillions, 1200 different species) researched to improve human health; probiotics, diet, etc Main source of SFA is the fermentation of non-digestible carbohydrates (dietary fibre) by gut microbiota (ex. propionate; C3, acetate; C2, and butyrate; C4) Metabolism of ethanol in liver (acetate) [draw] SCFA receptors mediate: Energy regulation Inflammatory responses Gut motility Appetite and glucose homeostasis So associated with obesity/T2D

Question 44

FFA2 and FFA3: natural and synthetic ligands

Answer 44

SCFA: propionate, acetate, butyrate Orthosteric and allosteric agonists and antagonists (ex. compound 6 is an allosteric antagonist for FFA3, and compound 1 is an orthosteric agonist for FFA2) CATPB is an orthosteric antagpnist fpr FFA2 Often specific for mice or humans Endogenous ligands for both have an overlap in their preferance: FFA2: C3/C4/C2 > C5 > C6 > C1 FFA3: C3/C4/C5 > C6 > C2 > C1

Question 45

FFA2 and FFA3 expression profile, signalling conjugated, activation effects

Answer 45

Both: immune cells, gut, pancreatic beta cells and adipose (although for FFA3 controversial) FFA2: Gq, Gi and Arrestin Highly expressed in monocytes and polymorphonuclear cells  FFA3: Gi Least well characterised GI TRACT FFA2: C2 (acetate) and C3 (propionate) stimulates GLP1 and PYY (reduces appetite) release from intestinal enteroendocrine cells FFA2 and FFA3 are in ghrelin containing cells in stomach and (primarily FFA2) inhibits ghrelin secretion INSULIN SECRETION In rodents: C2 activates FFA2 and enhances GSIS from mouse islets in vivo/vitro mice via Gq C3 activates FFA2 and FFA3 and inhibits GSIS via Gi (separate study) In humans: No effect of SCFA on GSIS However, SCFA do activate Gi and Gq INSULIN RESISTANCE WT: insulin resistance causes expansion of beta islet (to increase cell function) causing increase in FFA2 expression FFA2 KO: insulin resistance no longer expands beta cell mass so can't compensate for increased metabolic load ADIPOSE Similar to FFA4: FFA2 inhibits lipolysis (Gi; C2, C3 and C4), enhances adipogenesis (C2 and C3), upregulated in during differentiation (FFA2 siRNA inhibits; however in humans no relationship) INFLAMMATION: context dependent FFA2 (C2 and C4; Gi) promotes SCFA mediated chemotaxis of neutrophils Acetate inhibits secretion of TNFalpha from mononuclear cells ROS increases with C2 (FFA2; Gi and Gq) in immune cells, but decreases with C4 (FFA3; Gi) in colon

Question 46

Type 2 diabetes: prevalence, how it develops, risk

Answer 46

In 1996, 1.4mil diagnosed in UK, in 2019 now 3.8mil (over 90% type 2) Costs NHS £10bil on diabetes and diabetes related disease Develop insulin resistance in peripheral tissue (mainly liver; doesn't prevent gluconeogenesis, adipose tissue, skeletal muscle) preventing glucose uptake and energy storage Loss of pancreatic beta cell function More insulin produced to compensate and over time when this becomes inefficient patient is diagnosed as type 2 diabetes Characterised by high blood glucose level Obesity is around 80% of someone's risk for developing Type 2 diabetes - beta cells increase in size and increase more insulin to compensate for metabolic load. Higher BMI means increased load on beta cells and so more insulin produced

Question 47

Effects of insulin secretion and metabolic consequence of type 2 diabetes

Answer 47

Peripheral effects of insulin: most classical effects Skeletal muscle: promotion of glucose uptake via GLUT4 Liver: prevents gluconeogenesis, increases glycogen synthesis White adipose tissue: suppresses lipolysis and increases lipogenesis Central effects of insulin: through brain the peripheral effects are augmented Muscle and liver: whole body insulin sensitivity improved Adipose tissue: decreases lipolysis and increases storage Food intake: Suppresses appetite to reduce food intake Metabolic consequence of insulin resistance: Pancreas: hypersecretion of insulin then decreased in later insulin resistance with more beta cell apoptosis, less beta cell mass, hyperglucagonemia Adipocytes: more circulating fatty acids, hyperlipidemia Muscle: insulin resistance Liver: insulin resistance, more hepatic glucose output Gut: impaired incretin effect

Question 48

Difficulties studying SCFA receptors, how to bypass, and results

Answer 48

Differentiating the effects of FFA2 and FFA3 is difficult due to: Similar expression profiles (in a mouse model, FFA2 expression decreased with FFA3 KO) Very low potency (high concentrations of FA to activate and for FFA2/3 is difficult) Relatively similar pharmacology Bovine Preference for longer chain lengths to activate FFA2 - particularly sorbic acid; C6 which is 1000 fold more selective for bovine compared to human FFA2 Produce a receptor activated by a synthetic ligand and not the endogenous ligand: Receptor activated solely by synthetic ligand (RASSL) or Designer Receptor Exclusively Activated By Designer Drugs (DREADD)  FFA2-DREADD: Single C141G mutation in human FFA2 to make more potent for sorbic acid and to eliminate SCFA binding H242Q Retains high affinity for human specific antagonist ligands (ex. CATPB) to characterise inhibition of DREADD-FFA2 Study: Produce DREADD KI mice The physiological response from C3 is only due to FFA3 activation, and sorbic acid only FFA2-DREADD activation to differentiate roles Results: SCFA dependent regulation of lipolysis and GLP1 secretion is fully dependent on FFA2 activation, not FFA3 Novel info: FFA2 increases gut motility

Question 49

Challenges for the future developing new drugs for FFA receptors

Answer 49

Development of safer FFA receptor agonists -reduced liver toxicity (FFA1 ligands found to induce) Could the development of a potent FFA1/FFA4 dual agonist be more beneficial than a selective FFA1 or FFA4 agonist? FFA1 - involved in insulin secretion, glucose homeostasis and lipid metabolism FFA4 - anti-inflammatory effects (may mitigate FFA1 liver toxicity induced inflammation; hallmark of NAFLD), adipocyte differentiation, insulin sensitivity and lipid metabolism Combined the benefits of both and potential synergy Fully characterise arrestin-dependent FFA4 signalling Determine the function of the long form FFA4 receptor Further characterise the humanised FFA2 DREADD mouse model to determine FFA2 and FFA3 physiological effects Understand the relationship between gut microbes and FFA2/FFA3 signalling in relation to T2D

Question 50

Insulin: discovery, structure, signalling (draw), how its secreted

Answer 50

Discovered in 1921 One of most important discoveries in pharmacology Previously diabetes meant death Insulin: 51 aa Dimer of B and (shorter) A chain that are held together via interchain disulphide bonds Signals by binding the RTK insulin receptor (on target cells; liver, muscle adipose tissue) alpha subunits, causing dimerization, beta subunit autophosphorylation, leading to phosphorylation of downstream signalling - Insulin receptor substrate; IRS phosphorylation leads to stimulation of PI3K leading to phosphorylation of PDK1 then PKC (regulates glucose uptake, glycogen/lipid/protein synthesis), then phosphorylation of GSK3 (inhibits) mTOR (stimulates for protein synthesis), and JNK) Shc/Ras leads to phosphorylation of ERK1/2 and survival pathways Negative feedback loop of PKC, JNK, ERK1/2 phosphorylating S on IRS1, turning off Glucose transported into pancreatic beta cell via GLUT-2 (and GLUT-1 in humans) Glucose metabolization via glycolysis and citric acid cycle increases ATP/ADP ratio ATP dependent K+ channels close causing membrane depolarisation Ca2+ voltage dependent channels open, causing influx into beta cell Triggers exocytosis of stored insulin vesicles First phase of a rapid release of pre-stored insulin, then a slower, sustained release involving new synthesis and secretion Augmentation of insulin secretion (GSIS) through gastrointestinal hormones incretins - secretion of Glucagon-like peptide-1 (GLP1) in response to food intake and glucose dependent insulinotropic peptide (GIP) in response to glucose/fat intake, both to increase insulin secretion in a glucose dependent manner Ghrelin secretion: inhibits insulin secretion

Question 51

G beta/gamma signalling in diabetes

Answer 51

FFA1 Galphaq activate PLCbeta (catalyses PIP2 into DAG and IP3) IP3 induces release of Ca2+ from ER into cytoplasm Pancreas: Increases insulin exocytosis from granules Gbeta/gamma: activates PI3K gamma which remodels the cytoskeleton to aid with the fusion and transport of insulin granules to the PM

Question 52

Drug targets in Ubiquitination process and examples of a drug (3)

Answer 52

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