Question

You are a scientist at a pharmaceutical company and want to develop a new drug that can decrease heart rate. In order to do this you decided to target the beta 2 adrenergic receptor. You want to design a peptide that gets incorporated into the plasma membrane and interferes with beta 2 adrenergic receptor signaling.

1) Design the peptide sequence (10) and describe briefly each of the features and function of the sequences you incorporated (20). 5 sentences for each)

2) How would you test that the peptide is delivered to the plasma membrane ( 3 approaches), and that it is interfering with beta 2 adrenergic receptor signaling (3 approaches) (20). What are the advantages and disadvantages (3 each) of your approaches used (5). Maximum 10 sentences each.

3) How would you determine if the exocytosis (7) and endocytosis (7) of the beta 2 receptor is still occurring. What are the drawbacks of your approach (6). (10 sentences maximum for each)

4) How would you test if your peptide is folded correctly in the cells without having to determine its structure. (3 approaches) (10). How would you block these pathways that are activated specifically for your peptide (3 approaches) (10). What are the disadvantages and advantages of your method (3 each) (5). Maximum 10 sentences each.

Answer 1

Answering any 3 of the questions based on CHEGG rule:

2. The peptide sequence that would be employed would be the sequence of beta adrenergic receptor that can bind to a region where a specific adapter protein binds and prevent its receptor activation or a peptide sequence that can bind to an activated form of beta adrenergic receptor and prevent its signaling. The peptide can be fluorescently labelled and using fluorescence microscopy or flow cytometry one can see whether the peptide is delivered to the plasma membrane. For fluorescence based, advantage is the visualisation of the peptide on the cell mebrane. One can also quantify the fluorescen intensity based on certain softwares. flow cytometry can also quantify as to the level of fluorescence present on the cell. disadvantagesare that correponding positive and negative controls need to be used for the right conclusion. Also, one should be careful about using fluorescence as they may bleach with time. One can also employ other labels such as biotin and correspondingly detect using streptavidin to trace the movement of the peptide. Other method will be to isolate the membrane and see the presence of the peptide using a specific tagged antibody (say streptaviding HRP).For isolation of membrane, one needs to be careful in isolation and characterisation. Advantages include the ease of performing western blot and sensitivity of HRP,

3.Endocytosis can be determined by fluorescence microscopy, using specific beta adrenergic receptor antibody and corresponding secondary fluorescent antibody. One should also use a plasma membrane marker to look at co-localisation of the receptor with the membrane if required. The experiment will be one where overexpressing cells are untreated and other treated with the peptide. If endocytosis is not occuring the fluoresence in the plasma membrane will be more as compared to intracellular vesicular sites.

Similarly , if the exocytosis is blocked one should observe less fluoresence in extracellular region using microscopy in case of peptide treated cells. One can also obtain the cell supernatant and validate the exocytosis process is occuring using western blot.

Advantages/Disadvantages: For fluoresence microscopy/western blot, one needs to be careful about positive and negative controls.

4. One can first perform an in vitro circular dichrosm to look at its secondary structure. For ex vivo, one can look at the isolation of cytosolic fractions/cell supernatant and see whether the specific peptide binds to a matrix corresponding to a specific peptide antibody. If the structure is intact, it will bind and can give fluoresence. One can also employ computation methods (like molecular dynamics) to see whether the peptide folds well in an environment equivalent to a cell. One can also do co-immunoprecipitation to see whether both peptide as well as the protein both get picked up while blotting. Advantages/disdvantages: Both techniques are specific but both are slightly laborious though the matrix based method is a newly devised technique that is in demand.

To block a pathway, one needs to know first which pathways are activated/inhibited. Activated pathways can either be inhibited using small molecules or specific cell permeable inhibitors. Specific antibodies for particular receptors can also be used to inhibit the activated pathways.