Assessment - Data Analysis
For all questions illustrate your answers fully, describing what you did at every step and providing input/output illustrating what input/output was obtained.
You need to include, embedded, within your submitted work all relevant output from online servers, as appropriate, as well as a written dialogue to fully illustrate what work you carried out. For all parts describe how you obtained your data by stating the bioinformatics portal used and the search strategy.
Please quote accession numbers of all database files used.
Raloxifene is a drug used to lower the risk of breast cancer in women who are at a high or moderate risk of developing it and who have been through the menopause. In addition, Raloxifene can also be used to prevent and treat bone thinning (osteoporosis) in post-menopausal women. Raloxifene acts as a selective oestrogen receptor modulator (SERM) and acts like oestrogen by targeting the estrogen-receptor. In breast cancer cells the drug binds to the estrogen-receptor preventing the binding of estrogen which would ordinarily stimulate the cell to divide and grow. .
- Locate within the Protein Data Bank (PDB) the 3-D structure of a complex between Raloxifene and the estrogen-receptor. State what your chosen entry is, and download the coordinates for the structure to use with Rasmol to investigate your chosen structure.
- Using the program rasmol or Swiss-PDB Viewer produce an image of the protein that you think clearly illustrates the major structural features within the enzyme. State the commands used within the selected program to obtain your image.
- Using tools within the PDB investigate the interactions between the drug and its receptor. Illustrate with images the different types of interactions that exist and give details of these interactions in your discussion.
- Discuss which types of bioinformatics tools and programs could be used to design new potentially improved inhibitors for the estrogen-receptor.
Cytochrome P450s are a family of proteins involved in phase I drug metabolism reactions. They are highly expressed in the liver, in the endoplasmic reticulum membrane. In this question you will explore the use of protein-protein interaction databases to find out what other proteins P450s interact with and whether the potential partnerships could have biological significance.
- Use the UniProt file for human cytochrome P450 2E1 as your starting point. Summarise the key structural features of P450 2E1 including how it is able to bind to the ER membrane, and structural features of the active site.
- Use a range of PPI databases to identify possible protein partners. Summarise your findings.
- From your searches select three proteins with different activities that interact with P450 2E1, describe the evidence for the interaction and discuss whether these interactions could be relevant to P450's ability to metabolise drugs, in particular ethanol and paracetamol (acetaminophen). Wherever possible select proteins for which there is experimental evidence for the interaction.
Using the human sequence for Rhodopsin from Uniprot determine the domain(s) present within this protein sequence, using the Pfam domain database. State the domain(s) and the amino acid range for each domain.
Run homology modelling for this sequence using SWISS-MODEL to obtain a 3-dimensional structure for this sequence.
DISCUSS, in detail, the results of the modelling that you obtain, including an in-depth discussion of the model obtained, the template used by the program, and all of the key features of the model and its quality from the output generated.
Download the coordinates of the model obtained, as a protein databank (*.pdb) file, and create an image of your modelled structure using rasmol or Swiss-PdbViewer which clearly shows the main features of the model.
Micro-RNAs are known to target selected mRNAs as part of their mode of action. In this question you will explore the interaction between an miRNA and the mRNA of beta-site APP cleaving enzyme (BACE), and amyloid plaque protein (APP).
- Retrieve the sequences of human, mouse, dog and cow BACE1 and APP mRNAs. Align the 3' UTRs of each set of four mRNAs and identify on your output conserved sequence elements.
- Retrieve the file containing the sequence of human miR-15a from the miRNA database. Run the complete sequence of the miRNA on UNAfold and show the predicted structure of the RNA. Calculate the folding energy per base and comment on your findings.
- Using the mature sequence of miR-15a (this is given in the miRBase file) identify potential binding sites on the 3' UTRs of human APP and BACE1 mRNAs. Assume that the miRNAs will bind to complementary sequences in the mRNAs, but not necessarily with complete complementarity. You will have to use alignment software to map complementary regions. Describe the procedure you followed, discuss the output with reference to a diagram of the alignment.
- On the basis of your models how would you expect the miR-15a to affect expression of amyloid plaque protein?