BIOL3013 Ivo Tews: Protein Peptide interactions Lecture 1-5

What are the 4 distinct family of cytokines?

Tumour necrosis factor (TNF), Interferon, Chemokine, & Haematopoietin

What degrades HIF-1alpha in normal oxygen levels?

Prolyl hydroxylases hydroxylate HIF-1alpha which targets it for ubiquitination & degradation via the proteasome

What activates HIF-1alpha in low oxygen conditions

PHDs (prolyl hydroxylases) are inhibited & asparagin-hydroxylase hydroxylates HIF-1alpha which activates it for gene expression

When is erythropoietin (EPO) activated and what is it activated by?

Activated in low oxygen conditions by HIF-1alpha mediated gene activation

What does EPO do?

Stimulates RBC maturation & activates erythroid progenitor cells to increase red blood cell production

What anaemic diseases can EPO treat?

Chronic kidney disease, inflammatory bowel diseases (Crohn’s disease & ulcer colitis), & myelodysplasia (blood disorder where the bone marrow doesn’t produce enough healthy RBCs)

Definition of Haematocrit

Percentage by volume of RBCs in your blood

Why can cancer cause low haematocrit levels?

Cancer can cause the elevation of other blood cells like T lymphocytes which reduces the proportion of RBCs in the blood

Where is Epo produced & where does it act?

Produced in kidney interstitial fibroblasts & acts on erythroid progenitors (CFU-E) in bone marrow to stimulate red blood cell production.

Name the pathway of intracellular signal transduction of cytokine-cytokine receptor binding (like Epo-EpoR)

JAK-STAT pathway (JAK2-STAT5)= Janus kinase & Signal transducers and Activators of Transcription

Name the 2 important domains of JAKs

FERM domain: large N-terminus domain that binds the receptors’ Box1 motif- the main anchor point

Kinase domain: catalytically active domain that phosphorylates tyrosine residues on itself, the receptor, & STAT. Contains ATP-binding site & catalytic loop

Name the 3 important areas/domains of STAT

SH2 domain: mediates dimerisation & interaction with P-Tyr

DNA-binding domain: allows for regulation of gene expression by STAT

C-terminus: phosphorylated by JAK & interacts with SH2 domain on another STAT

Outline the steps for JAK-STAT pathway

Epo-EpoR binding→ Receptor dimerisation→ JAK cross-phosphorylation→ JAK activation & phosphorylation of receptor Tyr residues→ STAT binding to Tyr-P→ STAT phosphorylation by JAK→ Activated STAT release & dimerisation→ Exposure of NLS & nuclear translocation→ Activation of gene expression by DNA-binding domain

What is the short & long term regulator of JAK-STAT pathway?

Short term= SHP1

Long term= SOCS

How does SHP1 regulate cytokine signalling? How does it remain inactive under normal conditions

2 SH2 domains localise the protein to P-Tyr on receptor tail

Its phosphatase domain catalyses the inactivation of JAK by removal of P

Remains inactive by self-inhibition= self-folding onto itself where the SH2 domain interacts with its phosphatase domain

How does SOCS regulate cytokine signalling

SH2 domain binds phosphorylated residues of EpoR & JAK

SOCS box on C-terminus interacts with protein complex E3 ubiquitin ligase which ubiquitinates the receptor & JAK→ degradation by the proteasome

What is the definition of growth factors?

Protein that regulates cellular processes such as proliferation, differentiation & maturation

What are the events leading up to EGF ligand release & EGFR activation

Acetylcholine binds its GPCR (M1/M3) leading to the expression of ADAM, a membrane-bound MMP—> Cleavage of transmembrane domain of EGF allowing to to act in autocrine or paracrine manner on itself

What proteins are involved in the RTK→Ras activation cascade

EGF ligand, EGF receptor, adaptor GRB2 & SOS (Son of Sevenless)

List the steps from EGF-EGFR binding to Ras activation

RTK dimerisation & activation by cross-phosphorylation by intrinsic Tyrosine kinase activity→ Adaptor protein GRB2 recruitment by its SH2 domain, binds to P-Tyr residues on receptor→ SOS recruitment by GRB2’s SH3 domain→ Ras activation by SOS interaction with membrane bound inactive GDP-Ras—> Ras conformational change leading to removal of GDP & gain of GTP= active

What are the proteins in the MAP cascade (Ras)

Ras→Raf→Mek→MAPK

How is Raf kinase activated in the Ras pathway?

The N-terminal regulatory domain of Raf binds to GTP-bound Ras, removing the inhibitory 14-3-3 protein, allowing it to phosphorylate MEK

Similarities between EGF & EPO pathways

Both have signals that cross the membrane without the signalling molecule physically crossing the membrane; phosphorylation of both receptors creates docking sites for proteins with SH2 domains; and GRB2 & STAT both have SH2 domains;

What does GEFs, GAPs, RGS & GDIs stand for and are they activiating or inactivating proteins

GEF= Guanosine Exchange Factors (SOS), activating

GAPs= Guanosine Activating factors, inactivating

RGSs= Regulators of G-protein Signalling, inactivating

GDIs= Guanine nucleotide Dissociating Inhibitors, inhibits inactivation by GEFs

What key regions of SOS & Ras responsible for their interaction

SOS a-helix: responsible for displacing the bound GDP on Ras by binding the nucleotide-binding pocket

Switch I & II of Ras: regions that change shape depending on whether Ras is GTP or GDP bound. This change is crucial for Ras to interact with other proteins & trigger downstream signalling pathways (MAP cascade)

Equation for Kd

k_OFF/k_ON = [R][L]/[RL]

What is the technique used to find the Kd of receptors & their ligands

Surface plasmon resonance😊

What are the key components in surface plasmon resonance (SPR)?

Sensor chip: glass slide coated with gold, where the prey is immobilised and the resonance angle is measured

Prey: bound to sensor chip

Bait: molecules in the solution that flow over the sensor chip, unidirectional flow

Buffer solution: replaces the bait solution after association, required for dissociation of prey molecules, k_OFF

What are the negatives of surface plasmon resonance (SPR)?

Challenging to directly compare results= due to dependent variable variations between studies

Non-specific binding= bait molecules cans sometimes attach to the sensor chip, creating background noise & inaccurate readings

Differing results: results can vary with the same bait & prey if their roles are switched and the bait is immobilised on the sensor chip instead

What are the 2 types of interferons and what Jak/Stat dimers are used in the signal transduction process?

Type I= IFNa/b: via Jak1/Tyk2 & Stat1/2 heterodimers

• produced by DCs & fibroblasts for viral infection

Type II= IFN-gamma: via Jak1/2 & STAT1 heterodimers

• produced by T cells & NK cells

What does wound healing require (inflammatory wise)

Conversion of a pro-inflammatory response to an anti-inflammatory one. Done by switch in cytokine production from PDGF & G-CSF to IL-4 & IL-6 for example

What are the steps that occur prior to Myc activation

Mitogen activation: example is EGF to EGFR (RTK) leads to activation of Ras through adaptor protein GRB2 (SH2, SH3) & recruitment of SOS which removes GDP from Ras & is replaced with GTP= active Ras

MAP cascade: Raf→MEK→MAPK activation where it then translocates to the nucleus

Myc activation: MAPK phosphorylates Myc which activates it. It then acts as a transcription factor & induces gene expression for the cell cycle, including both Cyclins & CDKs

Define CDKs & Cyclins

CDKs= Cyclin-dependent kinases: regulate progression of the cell cycle by phosphorylation

Cyclins: protein family that act as regulatory subunits for CDKs, required for CDK activation

What are the 3 main proteins that control CDK activity

Wee1: deactivates CDK by phosphorylation at Tyr15

Cdc25: activates CDKs by de-phosphorylation at Tyr15

CAKs: activate CDKs by phosphorylation at Thr160/161 (depending on CDK)

Phosphorylation at Thr161 & dephosphorylated Tyr15 has to be present at same time for CDK activation, one or the other= no kinase activity

Steps of the G1 (R) & G1/S checkpoint

Myc activation leads to CDK activation→ Cyclin D-CDK4/6 phosphorylate Rb→ conformational change & release of TF E2F→ auto-stimulation & S-phase gene transcription→ Cyclin E-CDK2 activation→ Further Rb phosphorylation & progression into S phase with S-Cdk activation

How does DNA damage leads to p21^Cip1/Waf1 expression & what is its function?

DNA damage→ activation of ATM/ATR kinases→ activation of Chh1/Chh2 kinase activation→ p53 activation by phosphorylation & removal of its inhibitory regulator Mdm2→ gene expression of p21 (CDK inhibitor protein)→ binding & inactivation of G1/S-Cdk & S-Cdk complexes preventing S phase progression

What is the Raf recognition motif on Ras

DEYDPTIED

What is the 14-3-3 recognition motif on Raf

RSx(pS)xP

What do 14-3-3 proteins bind to?

Binds to the 2 phosphoserine residues on Raf

• 1st is on the N-terminal regulatory domain of Raf

• 2nd is on the C-terminal kinase domain of Raf

What is the structure of 14-3-3 proteins?

Homo/heterodimers (30kDa each monomer) that have phospho-binding moieties (positive residues)

Characteristic L-shaped structure formed by 9 alpha helices running in antiparallel manner

What are the overarching functions of 14-3-3 proteins (aside from binding Raf) & how is it similar to SH3 proteins

Cell division, apoptosis & differentiation

Similar to Sh3 proteins due to being a universal adaptor for phosphoserine where SH3 proteins are for phosphotyrosines

Where does the name 14-3-3 come from?

From its discovery in bovine brain homogenate resolved in starch-gel electrophoresis where it is the 14th fraction at position 3-3=

14-3-3

How were peptide libraries used to deduce the key residues in the 14-3-3 recognition motif of Raf

Used to deduce what residues had high values & therefore binding affinity by using all 19 amino acids to create 4.7×10⁷ unique peptides in a single synthesis

Found that proline at pSer+2 was vital for recognition & binding

What is unique about 14-3-3 proteins recognition motifs & how is it possible?

It can recognise & bind more than 1 motif, such as ARSHpSYPA & RLYHpSLPA. 1st one is cis-Pro & other is trans-Pro

What are NEF proteins?

Nef=negative factor proteins. Found on viruses & are vital for their entry, replication & exit of host cell

How is HIV-Nef similar to Ras GTPase?

HIV-Nef contains a peptide sequence that mimics that of active Ras GTPase when GTP-bound= contains 4 key residues xD_DPxxEx

How would HIV-Nef affect a host cell?

Would cause activation of Raf kinase, leading to the MAP cascade which causes growth & proliferation. This is favourable for viral replication & ext due to increased progeny release

How can EMSA (electromobility shift assay) be used to determine the HIV-Nef consensus sequence

Through fusion of NEF with GST (a solubility tag), this method utilises the interaction between certain amino acids & labelled Raf to determine binding (binding assay). By altering the amino acid chain the specific sequence motif can be found

What other sequence motifs does HIV-Nef contain and why is this abnormal?

o   ₁₅₄EE₁₅₅ : interacts with COP (coat outer protein)

o   ₁₆₀ExxxLL₁₆₅: interacts with AP1/2/3 (adaptor proteins in vesicular transport)

o   ₇₂PxxPxR₇₇: a SH3 recognition motif which interferes with T-cell receptor signalling (experimental data has shown this)

o   ₁₀₅RR₁₀₆: interacts with PAKs= kinase controlled by Rac/Cdc42 which play a major role in entry, replication & spread of viruses

o   ₅₇WL₅₈: interacts with CD4 causing its downregulation, avoiding superinfection= immune evasion

o   S₁₂₁FPD₁₂₄: interacts with thioesterase, required for viral endocytosis (entry)

₆₂EEEE₆₅: interacts with PACS1, a protein required for Golgi tracking

Abnormal because it contains so many different sequences that are recognised by so many factors, shows that it is picking up these motifs they prove to be useful so they remain, almost looks fake

How does Nef subvert host signalling

o   Regulating cell cycle control: stimulates MAP cascade by Raf interaction

o   Regulates host immunity: by CD4 downregulation avoiding superinfection, SH3 interaction interferes with TCR signalling

o   Regulate trafficking: AP1/2/3 signals regulate sorting into clathrin-coated pits, PACS1 interaction interferes with Golgi trafficking, thioesterase is needed for endocytosis, & PAKs are required for viral entry & replication intracellularly

What is the function of integrins?

They are signalling molecules that affect the activity of other cells. Typically this is in terms of communication & regulation of the immune system such as inflammation, and immune cell differentiation & proliferation