ZAP70 - A Potential Target for Immunosuppressive Therapy

Assignment Task

Background

ZAP70 is a cytoplasmic tyrosine kinase found mainly in T cells and is important both for T-cell development and for T-cell-receptor signalling (Figure). Its deficiency leads to severe combined immune disorder (SCID). ZAP70 is considered to be a relevant target for autoimmune disease, in which T-cell activation plays an important role. ZAP70 is also overexpressed in some individuals with chronic lymphocytic leukaemia, and these individuals have an especially poor prognosis. ZAP70 might therefore also be a useful therapeutic target in these patients.

In this practical, you will:

• Critically examine why the genetic code is degenerate
• Explore genome browsers and develop the skills for finding genes and proteins
• Examine different types of files for storing genetic information and their applications
• Learn the process for identifying protein drugs target and binding sites on proteins

Scenario

1. Genetic Code is Degenerate Although the DNA in the nucleus specifies the amino acids and their sequence, it is in the cytoplasm that the protein build-up takes place. The DNA of the nucleus makes a single strand of messenger RNA (ribonucleic acid) which leaves the nucleus and builds up the protein in the cytoplasm.

The proteins are essential for building cell structures and enzyme synthesis. Therefore DNA controls which enzymes are made and the enzymes determine what reactions take place; the structures and reactions in the cell determine what sort of a cell it is and what its function is. Hence, DNA exerts its control through the enzymes. The RNA code is complementary, but not identical, to the nuclear DNA. A group of three bases (a triplet) controls the production of a particular amino acid in the cytoplasm of the cell. The different amino acids and the order in which they are joined up determines the sort of protein being produced.

Figure 1 is a small, imaginary protein molecule showing how a sequence of 5 different amino acids could determine the shape and identity of the molecule. Each amino acid (Serine, Cysteine, Valine, Glycine and Alanine) is coded for by a particular triplet of bases. In this experiment, you will examine the genetic code and justify why the Codon is degenerate.

1. Using Figure

2, write out the codon for the protein in Figure

2. Convert the following DNA sequence: A-A-T-C-G-C-T-T-A-C-G-A, to mRNA sequence

3. Re-transcribe (i.e., write out the complementary bases) the RNA sequence to get your tRNA sequence

4. Break the tRNA sequence you found into three-base sets to get your anticodon

Question

a. What is anticodon?

b. What are the roles of anticodons?

Comparing

Comparing two or more things in biological data allows us to examine how closely related they might be, either in terms of function, evolution, or both. The most frequently used type of comparison in bioinformatics is sequence comparison to work out how closely related a nucleotide or protein sequence is to others in the public databases. This is done by aligning the sequences - rearranging them to find the best match possible - and takes into consideration insertions, deletions, and substitutions that may have occurred since divergence from a theoretical common ancestor. If a match is found we might be able to infer something about the relationship between sequences. We can perform pairwise sequence alignments and multiple sequence alignments; there are numerous different tools for performing such alignments, and the right one to use will vary depending on the context.

Controls

When it comes to comparing a sequence to entries in a sequence database (sometimes called sequence similarity searching) the challenge is in assessing whether a particular alignment is significant, not in the alignment itself. In this case, an alignment is significant when the likelihood of it occurring by chance (i.e. randomly) is small. This is expressed as the expectation score (also known as an e-value where the smaller the score, the more significant the alignment, and the more likely it is due to the existence of a shared ancestor and thus homology. Controls to check the validity of a sequence similarity search include comparing random sequences, and assessing the score of unrelated sequences.

Does youep ZAP70 have an active tyrosine kinase?

You are now working on a drug discovery programme to create new inhibitors of ZAP70 for use in autoimmune disease. Gina, a medicinal chemist in your group, has synthesised a series of protein tyrosine kinase inhibitors. You need to devise a cellular assay to see which ones inhibit the ZAP70 tyrosine kinase. You have ready access to primary T cells from the following animals: Mouse Rat Youep.

To decide which of your models has a ZAP70 protein most closely related to the human one, you have decided to do a quick multiple sequence alignment. Using EBI search, you located the canonical sequences (isoform 1) for the human, mouse, rat and youep ZAP70 proteins. For each one, you downloaded the protein sequence in FASTA format. You then performed a multiple sequence alignment of these proteins using the Clustal Omega tool.

On the basis of this alignment, you have decided to use sheep T cells for your assay but you want to double check that sheep ZAP70 has an active tyrosine kinase domain before wasting time on developing an assay. For this you’re going to use a tool called InterProScan. This tool compares your chosen sequence with all the sequences in InterPro, a data resource that provides functional analysis of protein sequences by classifying them into families and predicting the presence of domains and important sites.

Family: shows the family to which InterPro predicts the sequence belongs. Click on IPR012234 or PIRSF000604 to take you to the InterPro entry page for the family, where detailed information about its function may be found.

Domian: summarises the domain and repeats that InterPro predicts the protein to contain. The sequence is represented as a grey bar. Domains and repeats are indicated as coloured bars. Mousing over the bars reveals the domain ID, type of domain or repeat that they represent, along with their position on the sequence and a link to the relevant InterPro entry page. Clicking on the ID on the right side of the bar opens the InterPro Entry page with detailed information on the domain.

Unintegrated, Active Site, Homologous Superfamily, Binding Site: These sections hold detailed signature match information, showing the raw match position of all the different signatures in InterPro to the sequence, including (where available) signatures representing homologous families, domains, repeats, binding and active sites, and unintegrated signatures that are not associated with InterPro entries.

Gene Ontology (GO) terms section hold information GO predicted for the protein. These terms are assigned based on the matches to the InterPro entries. 10 obed.brew@uwl.ac.uk In summary the results show that sheep ZAP70 is indeed a member of the tyrosine protein kinase family, and that it has two SH2 domains and a protein tyrosine kinase domain. One thing to check is whether our sequence matches the patterns for the active site and ATP binding site, since any mutations in this area may make the protein enzymatically inactive. In this case, however, we can see that sheep ZAP70 contains the protein profile for both the ATPbinding site and the active site (Active Site). It should be safe to use sheep cells for the assay.

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