Lethal Mutation

The National Center for Biotechnology Information (NCBI) is a division of the National Library of Medicine (NLM) at the National Institutes of Health (NIH). The NCBI maintains many enormous databases, including GenBank, which stores all known public DNA sequences, submitted from individual scientists and large genome centers around the world; OMIM, a Web-based catalog that contains thousands of entries for genes and genetic disorders, which is frequently updated by NCBI staff based on published work in the scientific community; the Molecular Modeling Database (MMDB) of 3D protein structures; and many, many others. No other source of up-to-date molecular biology information is even remotely of the size and scope of this collection of resources.

Today, you will use the NCBI website (www.ncbi.nlm.nih.gov) to research a genetic disorder, ALS, and the mutant protein, SOD1, which causes the disorder. You will then download the protein structures of both the normal and two mutant SOD1 proteins and view them in 3D using downloadable viewing software called Cn3D. By the end of this activity, you should be able to describe the effect of the mutations in terms of the effect on protein structure and be able to correlate the molecular mutation to the physiological symptoms of the disease.

Activity Instructions:

Download the 3D protein viewer:

1. Click here to begin.

2. Click on “Download Cn3D 4.1 for PC, Mac, and Unix” and follow the instructions. (This will download the viewing software you will need to view a protein in 3D on your screen.)

Now you are ready to begin the activity:

3. Go to OMIM. Search OMIM for “SOD1.”

Your results: The first entry is *147450, Superoxide dismutase 1, SOD1 The second entry is #105400, Amyotrophic Lateral Sclerosis 1; ALS1

Start with the second entry first:

4. Click on the #105400 (second entry) to gather background information on ALS, the disease caused by mutant SOD. 5. Focus on Paragraphs 1,2, 10, and 14 to get an overview of key points.

6. Answer the following questions:

a. 15 to 20% of cases of familial amyotrophic lateral sclerosis type 1 (ALS1) are associated with a mutation in what gene?

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b. Familial forms of ALS follow what mode of transmission?

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c. What is another name for ALS?

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d. What is the nature of the symptoms and the disease progression?

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e. Does the disease show incomplete penetrance and/or variable expressivity?

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f. Why is euthanasia an issue involved with ALS?

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g. What is the title of the bestselling book by Mitch Albom, describing the last year in the life of an ALS patient?

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7. Use the back arrow button to return to the OMIM results list. 8. Review entry *147450 (regarding SOD1) paragraphs 3, 13, and 16 and answer these questions:

a. What reaction is catalyzed by superoxide dismutase?

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b. What scientist identified the first disease-producing mutations in SOD1?

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c. What type of mutations were found?

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d. Describe the two proposed mechanisms by which mutant SOD1 would cause cellular damage in ALS patients.

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e. What specific mutation was found to be most frequent?

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f. Examination of the crystal structure of human SOD established what?

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9. Click on “Links” for SOD1 (to the far right), and scroll down to “Structure” and click.

10.This takes us to the structure database. We will be using the following entries:

1UXM A4V mutation of human SOD1

1UXL I113T mutation of human SOD1

1SPD Human superoxide dismutase 1 (SOD1)

11.First click on 1SPD, the normal structure of SOD1, and on the next screen click on “View 3D Structure” (the gray button).

The viewing program should open up a new window with the structure of SOD1. Note that you can click and drag on the image to rotate it in 3D. 12.Answer the following questions:

a. What secondary structures are visible? How many can you find of each type?

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b. What metal cofactors are present?

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c. Locate the amino and carboxyl termini. What does this tell you about quaternary structure?

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13.Now click on 1UXM, view the structure and answer these questions:

a. Which level of protein structure is visibly altered by the A4V mutation, compared to the normal SOD1 structure?

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14.Now click on 1UXL, study the structure and answer these questions:

a. 1UXM is described as an “A4V mutation of human SOD1.” This means that the fourth amino acid in the protein has been changed from adenine to valine, due to a missense mutation in the gene. 1UXL is described as an “I113T mutation of human SOD1.” What does this mean?

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b. Which level of protein structure is visibly altered by the I113T mutation, compared to the normal SOD1 structure?

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15.Close the structure viewing windows and use the back arrow to return to the structures list. On the right side of the 1UXM entry, click on “Links” and drag down to “Full text in PMC.” This will take you to a list of 2 journal articles which study the A4V and I113T mutations. Click on the “Abstract” of each article, read through them quickly, and answer these questions:

a. What can you find out about the location of the A4V and I113T mutations, in terms of both tertiary and quaternary structure?

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b. What effect do these mutations have on the dimer formation?

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16. Homework (check with your instructor for due date): Use your answers to all the previous questions in this activity to synthesize a 1-page written summary describing how mutation of the SOD1 gene relates to the disease ALS. The summary should include a description of ALS on both a physiological and cellular level and the mode of transmission in familial cases; the normal structure and function of SOD1 protein in a motor neuron; the types of SOD1 mutations associated with ALS; and an explanation of how these mutations affect the structure of SOD1 and thus cause neuronal death. The summary should be 3/4 page typed, 250-300 words.

Referenced URLs:  www.ncbi.nlm.nih.gov