Aspartic proteinase enzymes are a family of enzymes involved in a number of important biological processes. In animals the enzyme renin has a hypertensive action through its role in the renin-angiotensin system. The retroviral aspartic proteinases, such as the HIV proteinase, are essential for maturation of the virus particle and inhibitors have a proven therapeutic record in the treatment of AIDS. The lysosomal aspartic proteinase cathepsin D has been implicated in tumorigenesis and the stomach enzyme pepsin, which plays a major physiological role in hydrolysis of acid-denatured proteins, is responsible for much of the tissue damage in peptic ulcer disease. Since aspartic proteinases also play major roles in amyloid disease, malaria and common fungal infections such as candidiasis, inhibitors to these enzymes are much sought after as potential therapeutic agents.
• Locate within the Protein Data Bank the 3-D structure of an aspartic proteinase enzyme. State what your chosen entry is, and download the coordinates for the structure to use with Rasmol to investigate your chosen structure.
• Identify, using an appropriate bioinformatics program, the active site residues for your chosen PDB entry. Show the output generated by the program used.
• Using the program rasmol or Swiss-PDB Viewer produce an image of the structure that you think clearly illustrates the major structural features within the enzyme and clearly shows the location of the active site residues. State the commands used within the selected program to obtain your image.
• Discuss which types of bioinformatics tools that could be used to design inhibitors for aspartic proteinase.
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 2D6 as your starting point. Summarise the key structural features of P450 2D6 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 2D6, describe the evidence for the interaction and discuss whether these interactions could be relevant to P450’s ability to metabolise drugs. Wherever possible select proteins for which there is experimental evidence for the interaction.
Using the human sequence for the 509 amino acid protein Tyrosine-protein kinase Lck (Uniprot sequence entry P06239) determine the domain present within this protein sequence, using the Pfam domain database. State the domain and the amino acids within the 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 models obtained, the templates used by the program, and the output generated.
Download the coordinates of what you consider to be the ‘best’ 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.
In lecture 6 on RNA informatics you were shown an analysis of RNA structure across the 3′ UTR of interleukin 2 (IL-2). Many other cytokines also carry an AU-rich element sequence, including interleukin 6 (IL-6). Micro-RNAs are known to target some cytokine mRNAs. In this question you will explore the interaction between the mRNA of IL-6 and a micro-RNA miR-365. It has been shown that miR-365 inhibits expression of IL-6 through this interaction.
a) Retrieve the sequence files for human IL-6 and five other species. Align the 3′ UTRs of the six mRNAs and identify on your output potential AU-rich sequence elements.
b) Retrieve the two files containing the sequence of miR-365 from the miRNA database www.mirbase.org. Run the complete sequences of the RNAs on Mfold and show the predicted structure of the RNAs. Calculate the folding energy per base and comment on your findings.
c) Model the binding of the mature sequence of each miRNA (this is given in the miRBase file) with the 3′ UTR of IL-6 mRNA. Assume that the miRNAs will bind to complementary sequences in the IL-6 mRNA, 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.
d) On the basis of your models predict which miRNA would be inhibitory and explain why. Does the mechanism involve the AU-rich element of IL-6?