Project Manual Bio3055. Lung Cancer: Cytochrome P450 1A1

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1 Project Manual Bio3055 Lung Cancer: Cytochrome P450 1A1 Bednarski 2003 Funded by HHMI

2 Lung Cancer: Cytochrome P450 1A1 Introduction: It is well known that smoking leads to an increased risk for lung cancer, but how does genetics play into the risk for developing lung cancer? The transformation of a normal cell into a cancerous cell can result from many causes. In one model, factors that lead to an increased rate of mutation in DNA increases the chances that a proto-oncogene will be mutated into an oncogene, causing a normal cell to be transformed into a cancerous cell. For your project, you will examine one gene that has been proposed to be a risk factor for lung cancer. Some research has shown an increased incidence for lung cancer in smokers who have a particular polymorphic version of the gene for cytochrome P450 1A1 that is expressed in the cells that line their lungs. Cytochrome P450 1A1 is an enzyme that oxidizes polycyclic aromatic hydrocarbons (PAHs). The basic mechanism that has been proposed is that this enzyme converts aromatic hydrocarbons in cigarette smoke into more toxic compounds that can modify DNA and that this leads to a higher rate of mutation in the cells that line the lungs. In this module, you will use web-based bioinformatic tools to investigate the molecular differences between the wild type and a polymorphic version of cytochrome P450 1A1. You will be studying the cdna sequence that codes for CYP1A1 with a mutation. You can obtain this sequence on the course website under your project heading. The file name is shown below: Saved in FASTA format in file CYPmutseq For your research project, you will analyze the mutant P450 1A1 protein using the bioinformatics tools presented in lab. You will investigate the structure of the P450 1A1 protein, model the mutation, and find out what is known, if anything, about the biological impact of the mutation. Through your studies, you will form a hypothesis about what the structural and biological effects are of this mutation, and organize the results of your research into a report. At the last lab session you will present your report to a small group. Laboratory 1 No Pre-lab assignment Tutorial on web-based tools Laboratory 2 Pre-lab assignment: 2

3 Complete Reading Questions 1 (page 5). In this reading, the relationship between environmental and genetic risk factors for cancer are discussed. You can find this article on the course website under your project heading. Answer the questions for reading 1 in your project packet and turn in your written answers at the second lab meeting. Readings 1 Perera, F.P., Uncovering New Clues to Cancer, Scientific American, p (May 1996). Laboratory 3 Pre-lab assignment: Complete Structure Problem Set (page 7). Complete Reading Questions 2 (page 6). This reading provides you with some background for working with the crystal structure of a cytochrome P450 enzyme. The abstract shown below summarizes how expression of cytochrome P450 enzymes in lung tissue is related to lung cancer. The excerpt is for the article that contains the crystal structure you will be studying. Both of these readings can be downloaded from the course website under your project packet heading. Answer the questions for reading 2 in your project packet and turn in your written answers at the third lab meeting. Readings 2 Abstract from: Hukkanen, J., et al, Expression and regulation of P450 enzymes in human lung, Crit Rev Toxicol., 32, p (2002). Excerpt from: Williams, P.A., et al., Crystal structure of human cytochrome P450 2C9 with bound warfarin, Nature, 424, pp. 464 (2003). Laboratory 4 No Pre-lab assignment If you haven t yet, you should begin preparation for your final report. Laboratory 5 Pre-lab assignment: For this lab, you need to assemble all your research into a report format so you are ready to present your results to the other group working on lung cancer. The other group you will be meeting with has been researching the protooncogene, K-Ras. Follow the format given in your lab manual for writing the report. At the last lab meeting, you will have 20 minutes to present your findings 3

4 to the other group. Then they will present their findings. The rest of the lab will be spent working as a group to provide answers to a joint quiz. You will then hand in your reports to be graded. 4

5 Questions on Reading 1 Cytochrome P450 1A1 Scientific American article, Uncovering new clues to cancer risk 1. What does PAH stand for? 2. Name two sources for PAHs in our environment. 3. Explain how PAH-DNA adducts can lead to cancer. 4. Explain how certain forms of P450 1A1 may increase cancer risk. 5. List one more Biomarker for Susceptibility besides P450 1A1 and explain how inheriting that mutation can lead to cancer. 5

6 Questions for Reading 2 Cytochrome P450 1A1 Excerpt from Nature paper: 1. What types of compounds do cytochrome P450 enzymes metabolize? 2. Which CYP isoform was crystallized for this paper and what organism is it from? 3. How has the proposed role of Arg 97 changed due to the solving of this crystal structure? Abstract from Crit Rev Toxicol paper: 4. Expression of CYP enzymes in what tissue type is discussed in this review? 5. What do the authors propose could lead to differing risks for lung cancer for different individuals? 6. What isoforms of P450 enzymes were found to be expressed in lung tissue? 6

7 Structure Problem Set Directions Draw the chemical structures for the following amino acids. They are represented in cpk color mode (see Glossary for more information)

8 4. Draw the chemical representation of the following tripeptide. 5. Draw the chemical representation and represent H-bonds as dotted lines between the atoms where distances have been measured. You will need to add hydrogens that don t appear in the picture below. 6. What distance must two atoms be in order to be involved in hydrogen bonds and ionic bonds (use the Berg textbook, p if needed)? 8

9 Guide Sheet 1 Hints and Tips for P450 1A1 Translating the sequence Obtain your patient s cdna sequence from the course website (Cypmutseq) Use Reading Frame 3 when translating the sequence at the Sequence Manipulation Suite. NCBI Gene Using Gene, find the entry for CYP1A1. Select the Homo sapiens entry from the search results. Answer question 1. Swiss-Prot Entry Use cyp1a1 to search the SwissProt database and be sure to select the human protein from the search results. Answer questions 2 5. BLAST and ClustalW Be sure to choose a good variety of sequences from the BLAST search. The more varied the sequences, the more interesting the alignment will be to study. Be sure the wild type human (RefSeq) and mutant sequences only differ by one amino acid residue. If more differences are found, there may have been a mistake in the translation of the mutant sequence. Answer questions

10 Cytochrome P450 1A1 Questions to accompany guide sheet 1 Locus Link Entry 1. Fill in the following information from the Gene entry: a. Write the GeneID number here. b. What is the gene name? c. Where in the human genome is this gene located? d. What is the RefSeq number for the mrna sequence? e. What is the RefSeq number for the protein sequence? Swiss-Prot Entry 2. What tissues express this protein (under comments )? 3. What is the chemical reaction catalyzed by this enzyme? 4. Where in the cell is this protein found? 5. Which amino acid residue number binds the iron of the heme (under Features )? 10

11 Multiple Sequence Alignment 6. What is the mutation? Write it in the following format Res123Res where the first Res is the three-letter code for the amino acid in the un-mutated (wild type) protein and the second Res is the amino acid in the mutated protein. In place of 123 put the amino acid residue number of the mutation. 7. Is the mutation in a region of conservation? 8. What is the secondary structure predicted for the region containing the mutation? 9. What type of secondary structure(s) is the most common in the alignment? If it seems to be an even mix between alpha helices and beta sheets, state that. 11

12 Guide Sheet 2 Hints and Tips for P450 1A1 Searching for Structure Files: Since the crystal structure of cytochrome P450 1A1 has not yet been solved, you will analyze the crystal structure of a closely related protein, cytochrome P450 2C9. This is the structure you read about in the reading assignment due for this lab. To obtain the crystal structure data file (pdb file), follow these steps: 1. Go to the Protein Data Bank website (see Glossary) which contains all of the macromolecule 3-D structure files (pdb files). Pdb files are named in 4 characters (numbers and letters). a. Search for the 1OG5 pdb file. The summary information page for 1OG5 contains a title for the entry, the compound crystallized, and the species of the source of the protein. Use this entry to answer questions 1-3. a. Click on Download/Display file at the left of the screen. b. On this page, choose to download the structure file in PDB format with no compression. It will be the none option in the second table. The 1OG5.pdb file should now be on your desktop. Viewing the structure file: IMPORTANT: The crystal structure contains a dimer of P450 2C9 with a bound substrate, warfarin (which is an example of a polycyclic aromatic hydrocarbon PAH). The two chains of the dimers are called, Chain A and B. The substrate is labeled, SWF and the heme is labeled, HEC in the control panel. 2. Swiss-Pdb Viewer/DeepView has been loaded on your desktop. To open 1OG5.pdb in this program, drag the file to the Swiss-Pdb Viewer/DeepView icon and drop it on the icon. In some cases, doubleclicking on the file will also open the pdb file in DeepView. 3. A black screen should appear with the protein shown in wire form. This is a difficult form to view the protein, so we are going to change it to the ribbon form mode. To do this, follow these steps: a. First make sure the control panel is open. If you don t see it, select control panel under Wind b. Click on the control panel window. You can see that all the amino acid residues in the protein are listed in the first column by 3-letter code and residue number. The next columns allow you to change what is displayed. In order to clean up the display of the enzyme, follow these steps: 12

13 c. Erase all the check marks in the show column and the side (meaning side chain) column by clicking on them. For now, you will only view the protein backbone in a ribbon diagram. d. Put check marks in the ribbon column for all the residues in the Chain A (stop when you see Chain B residues). For now, it is easier to just view one polypeptide chain. e. Locate the warfarin (SWF) and the heme (HEC) for the A chain in the control panel. These will be at the end of chain B in the control panel. Put checks in the show column so these molecules will appear in the display. The warfarin and heme will appear in the CPK coloring mode which is based on atom type: red = oxygen blue = nitrogen orange = phosphorous yellow = sulfur and phosphorous gray = carbon light blue = hydrogen f. Go to the main window and click on the Display menu and select Render in Solid 3-D. You should now be viewing a ribbon diagram of your protein. g. You can change the ribbon colors to any color you think looks best by selecting ribbon under Prefs. In this window, make sure the render as solid ribbon option (near the top) is selected. You can select different colors for the top, side, and bottom of the ribbons. This allows you to choose a darker version of the same color for the bottom of the ribbon to enhance the 3-D viewing. Take a minute to play around with this option and to color your protein the way you want. You can also change the background to any color by choosing Colors under Prefs, then background. h. Click in the display window to make sure that window is the active one. The tool bar for this window is located at the top and is described in your lab manual. Select the rotate tool. To rotate the protein, click and hold on the picture while moving the mouse. The other two buttons are zoom and transverse for zooming in on the protein and for moving the protein from side to side across the screen. Answer questions 4 and 5. i. Once you have a view that you like of your protein, save it by going to File then Save. Then select Layer. Name your file cyp2c9.pdb and save to desktop. When you open this file, all your colors and the orientation should by saved, but you will have to select Render in Solid 3D again under Display to see it. 13

14 Printing the 3D Figure of Your Protein 4. To save the pdb file as a photo file, we will use the program Grab. You can open Grab by clicking on the scissors icon in the toolbar of your desktop. 5. Make sure the figure is visible exactly the way you want it in Swiss-Pdb Viewer. Then, in Grab, go to Capture then Selection. You can now draw a box around the part of the view in Swiss-Pdb Viewer that you want to save. Save the file as cyp1.tiff. Save it to your desktop. You can use this same method to create a protein structure figure for a PowerPoint presentation. 6. Open the.tiff file in Preview. Choose Page Setup under File and change the scale to 70% to make sure the figure prints on one page. Print a copy of your.tiff file. Viewing an amino acid side chain 7. The residue corresponding to the residue that is mutated in your patient s protein is Leu440 in this structure. Show this side chain by clicking on the show and side columns in the control panel for that amino acid. Zoom in on this amino acid. Rotate the structure until you can get a good view of the leucine side chain. At this point, you can erase the check marks in the show column for all the other residues in order to focus on this side chain, the heme molecule, and the warfarin. 8. Confirm the identity the Leu side chain by using the identity tool on the toolbar and clicking on the side chain in the display window. Investigating the environment of the side chain In order to investigate the non-covalent interactions of the side chain, use the distance tool on the toolbar to find atoms that are close enough to atoms in the side chain to be involved in H-bonds, ionic bonds, or Van der Waal s interactions. Keep in mind the atom type when determining what type of interactions may be occurring. The heme molecule will be the focus for identifying non-covalent interactions of the leucine side chain. 9. Use the distance tool to measure the distances between an atom of the leucine side chain and an atom of the heme molecule. Measure the distances between the atoms that appear closest in the leucine side chain and the heme molecule. You will click on the two atoms that you want to 14

15 measure the distance between, then the distance, in angstroms, should appear.. This step may take several attempts. If you need to erase distances or labels, you can go under Display to labels, then select erase user labels. Answer question 6. Keep in mind the resolution of the crystal structure provides the error in the distances that you are measuring. For example, if the distance is 5 angstroms and the resolution is 2 angstroms, the distance between the atoms is estimated to be 5 angstroms ± 2 angstroms. 10. Print this view with the distances using the same method as in steps 4-6. Save this view as Leu.tiff Modelling the Mutation 11. To change the leucine side chain to a different side chain, use the mutate tool on the toolbar. Select the amino acid valine (Val) to mimic your patient s mutation. The Val side chain will appear in the lowest energy conformation (most stable). Some strange green lines may appear which represent potential H-bonds. You can make these disappear under Display, then deselect Show H-bonds. 12. Use the distance tool to measure the distance between the Val side chain and the heme as you did in step 9 with leucine. You will click on the two atoms that you want to measure the distance between, then the distance, in angstroms, should appear. Save and print this view following steps 4-6 and naming the file, Val.tiff. Answer questions 7 and 8. To put in report: For this lab, you will need the three figures printed in steps 6, 10, and 12. Make sure the residue numbers and distances are labelled. The distances and labels can be added by hand to the figure if they are difficult to see in the print-out. 15

16 Questions to Accompany Guide Sheet 2 P450 1A1 1. What organism was P450 2C9 obtained from for the crystal structure? 2. What is written for the Description line of the entry? 3. What is the resolution of this crystal structure and what does resolution mean for a crystal structure? 4. Carefully examine the secondary structure in the crystal structure and record any positions where the PSIPRED predictions were incorrect. Keep in mind that the this is still an estimate since the crystal structure you are examining is P450 2C9, not P450 1A1. 5. PSIPRED states its predictions are ~80% correct. Do you agree this is a good estimate of the accuracy? 16

17 6. Draw the Leu440 side chain and the closest part of the heme molecule from the crystal structure. Include the distances between the atoms you measured. This should include the correct chemical structure of the leucine side chain including atom types, the correct placement of hydrogens, etc. 7. Analyze the model of the Val mutation. Can the same non-covalent interaction(s) occur with Val and Leu? Draw a chemical representation of the Val residue and the atom(s) of the heme molecule that are closest to the Val. Show any changed non-covalent interactions and distances measured between atoms. 8. What is your hypothesis for how the valine mutation affects the activity of P450 1A1? Keep in mind that enzyme acitivity can change due to changes in the position of the heme (remember the R and T states of hemoglobin). Do you hypothesize the mutant P450 1A1 to be more active, less active or no change from wild type P450 1A1? 17

18 Guide Sheet 3 Hints and Tips for P450 1A1 OMIM search Search the OMIM database for CYP1A1. Click on the search result that states this exact gene name. Read the first paragraph for this entry and answer question 1. Select the Peterson reference in the first paragraph. This will take you to the citation information for that journal article. Now click on the PMID# to read the abstract for the article. Answer questions 2 5. Return to the OMIM entry for CYP1A1 and scroll down until you reach the paragraph that begins with Polycyclic aromatic hydrocarbons (PAH s). Read this paragraph and answer questions KEGG pathway Go to the Gene entry for human CYP1A1. Scroll down to the General gene information section and select the link for the KEGG pathway Tryptophan metabolism. You should see a nice graphic for the proteins in the pathway. The enzymes can be identified by their EC numbers. The EC number for CYP1A1 is Answer questions

19 Questions to accompany guide sheet 3 P450 1A1 1. What did the Kouri article in 1982 find about CYP1A1 and lung cancer? 2. What journal was this article published in? 3. At what institution did these authors work? 4. What compound do they call a procarcinogen? 5. How is CYP1A1 related to the procarcinogen? 6. What percentage of Caucasians has the highly inducible form of CYP1A1? 7. What type of smoker is most susceptible to an increase risk for lung cancer due to inheriting the CYP1A1 leu to val polymorphism? 19

20 8. What is the proposed mechanism for the increased cancer risk for people who have the valine-type CYP1A1 enzyme? 9. What is the difference between highly-inducible and enhanced catalytic activity for an enzyme? 10. How are the levels of PAH-DNA adducts affected by the exon 7 leu to val polymorphism? 11. What KEGG pathways are CYP1A1 a part of? 12. Draw the reaction catalyzed by CYP1A1 in the KEGG pathway: Tryptophan metabolism. Include the structures of the substrate and product. 20

Project Manual Bio3055. Lung Cancer: K-Ras 2

Project Manual Bio3055. Lung Cancer: K-Ras 2 Project Manual Bio3055 Lung Cancer: K-Ras 2 Bednarski 2003 Funded by HHMI Lung Cancer: K-Ras2 Introduction: It is well known that smoking leads to an increased risk for lung cancer, but how does genetics