Lecture 6. Engineering Signalling Pathways Flashcards
What did engineering chemoreceptor specificity in E. coli allow?
E. coli cells labelled with GFP, see which chemoattractants induced movement of the cell
Whether you could change the specificity of 4 alpha helices to introduce alternative types of attractive molecule (aside from aspartate, changed to argenine)
What is the role of chemical biosensor systems in yeast?
Pheromone sensing system in yeast, allows haploid yeast cells to detect pheromone peptide that is synthesised and released by other haploid cells.
Basis for asexual reproduction cycle in yeast.
When the mating factor interacts with GPCR, triggers pheromone system.
Final step is activation of transcription factor that turns on specific genes for mating with other mating factor type yeast cells, fusion to form diploid cells. Can be hijacked by changing the wild type GPCR of yeast. Used for generation of fatty acids
Why are some GPCRs replaced in yeast cells by mammalian GPCR?
Some are sensitive to medium chain fatty acids, so can monitor production over time, can see what strains have best production
How can a yeast fungal pathogen biosensor be created?
Replace Ste2 (detects yeast) with appropriate homologues from other fungal organisms (such as pathogenic organisms) - need reporter to detect whether pathogenic organism is there
Binding of pathogenic organism and mating peptides switches on lycopene production
What is the difference between the signalling pathways of prokaryotic and eukaryotic systems?
Prokaryotic: pyruvate-sensing network
Eukaryotic: cell migration
What happens when a GPCR binds to an agonist ligand?
Conformational change allows heterotrimeric G-protein to split into alpha and beta-gamma subunits, GDP replaced by GTP, initialising signalling cascade
How many GPCRs are encoded by the human genome?
800 GPCRs encoded in the human genome
400 dedicated to olfaction (sense of smell)
What eukaryotic organisms do not have GPCR pathways?
Plants
What powerful physiological responses in yeast and mammals do GPCR regulate?
Yeast: mating pheromone response
Mammals: flight or fight response
What proportion of all prescription drugs target GPCRs?
30-50% of all prescription drugs
For example, β-blockers to treat high blood pressure
What are examples of illicit drugs that target GPCR?
Cannabis (cannabinoid receptor); heroin (opioid receptor); LSD (serotonin receptor)
What is the purpose of DREADDs?
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)
Trying to modify specific receptors responsible for triggering certain pathways
What is the function of the human M3 muscarinic receptor?
Expressed in parasympathetic nervous system
Activated by the neurotransmitter acetylcholine (ACh)
What did DREADD do the human M3 muscarinic receptor?
Mutated receptor so that it wasn’t activated by Ach, but the synthetic ligand (CNO)
What is special about clozapine-N-oxide (CNO)?
It’s pharmacologically inert (invisible to other processes)
What have DREADDs/CNO been used for?
Regulate neurones responsible for food intake
Regulate neurones responsible for sleep and circadian rhythms
What are the potential uses of DREADDs in the future?
Express DREADD in serotonergic neurons in the brainstem
Use CNO to activate neuronal circuits implicated in depression
Reduced side effects compared with antidepressants
What is refactoring?
Refactoring means redesigning/restructuring a system in a way that maintains that system’s functionality while simplifying it to facilitate understanding and modification
What is kinetic modelling?
Basic synthetic systems can often be described by simple binding equations
More complex systems require more advanced mathematical models.
Why is kinetic modelling used for GPCR?
GPCR pathways have been progressively modelled since their discovery
GPCR models have become more complex to reflect experimental observations.
However, they are still too simplistic to reflect the complexity of signalling in vivo
How was the challenge of quantitatively modelling GPCR signalling overcome?
Deleted 11/15 genes from the yeast mating and glucose-sensing pathways (Only core MAPK relay elements retained)
Minimal set of components reintroduced into a refactored pathway (insulated pathway and tuneable components)
How can you engineer a mammalian photo-regulatable motility control system?
Rac1 is a GTPase involved in regulating actin cytoskeletal dynamics in metazoan cells
Light switch is engineered fusion that enables the application of light to trigger a conformational change that effectively dissociated Rac1 LOV interaction. That blocks the effector that is a kinase, use mutant form that is photoactivatable, this results in the activation of motility. Rac1 part of signalling pathway tied to motility. Fusion protein controlling the effector kinase. Light activates conformational change, allowing photoactivatable movement
