Taster Protocol

Protein interactions are central to many biological processes and there are many online databases with information about proteins including the NCBI and Uniprot sites. Scientists have a sense of humour and this can be reflected in the naming of some proteins. For this taster the target protein being looked at is has the same name as a well-known Simpson's character - Homer. This protein is found in the brain and has been suggested to play a role in stress, a very appropriate role for a compound named after Homer Simpson!

To look at proteins that might interact with Homer a method using the
mRNA for Homer from the NCBI database will be used to generate an antisense peptide that is then used to search within a protein database. Proteins with similarity to the antisense protein should bind to the homer protein and potentially have biological effects.

With a Biomedical Science slant the protein databases to be searched will be from the the bacteria that causes the disease Listeriosis (
Listeria monocytogenes), a serious infection usually caused by eating contaminated food.

HomerList1


The methods detailed below are aimed at secondary school and college students as a taster to the full version detailed on this website. The method is based around antisense peptide screening to identify interactions between a target protein (in this case Homer) and other proteins (in this case from the bacteria - Listeria monocytogenes).

Method:

  • Step 1: Click on the link for the Taster Script, which will open in a new window, and copy the full text displayed. Then return to this window.

  • Step 2: Using the link for the Python Compiler, which will again open in a new window, paste the copied text from Step 1 into the compiler. Then return to this window.

  • Step 3: Click on the link to the Homer mRNA, which will open in a new window, and copy the full text displayed. Then return to this window.

  • Step 4: Return to the open Python compiler window, click the play button, type in the Homer followed by a return where it asks for a name and then paste in the text from Step 3 where it states mRNA. Copy the complete Antisense sequence generated, starting with a Y. Then return to this window.

  • Step 5: Click on the link to the protein BLAST program, which will open in a new window and paste the antisense sequence into the "Enter Query Sequence" box. Then return to this window.

  • Step 6: Adjusting the settings to perform the protein database search. Return to the open BLAST program window.

  • (a) In the "Job Title" box type Homer.

  • (b) In the "Choose Search Set" section make sure that "Standard databases (nr etc.): is selected.

  • (c) In the "Standard" section "Database" box select "RefSeq Select Proteins (refseq_select)" from the dropdown link.

  • (d) In the "Standard" section "Organism" box type "Listeria monocytogenes (taxid:1639)".

  • (e) In the "Standard" section "Exclude" section click the "Models (XM/XP)" and "Uncultured/environmental sample sequences" boxes.

  • BLAST1List

  • Step 7: Adjusting the Algorithm settings. Return to the open BLAST program window.

  • (a) Click the "+ Algorithm parameters" title, this will change to "- Algorithm parameters" and display some further options.

  • (b) In the "General Parameters" section "Expect threshold" change this to "10"

  • (c) Click the "BLAST" button.

  • BLAST2List

  • Step 8: When the results appear you will see the names of the Listeria monocytogenes proteins that Homer interacts with. The third and forth of these (UDP-N-acetylmuramate dehydrogenase) are a protein involved in the manufacture of the bacterial cell wall that Homer could target to try and destroy the bacteria!

  • Results1



HomerListeriaTarget


Model of Homo sapiens Homer protein residues 122-134 (red) bound to Listeria monocytogenes UDP-N-acetylmuramate dehydrogenase residues 213-226 (blue) as predicted from antisense results generated is Steps 1-8 above, model generated using ClusPro.


HomerListeriaFull


Model of full length Homo sapiens Homer protein (red) bound to full length Listeria monocytogenes UDP-N-acetylmuramate dehydrogenase (blue) generated using ClusPro.


The diagrams above represent 3D models of the interaction between the Human Homer protein and the
Listeria monocytogenes UDP-N-acetylmuramate dehydrogenase. They were created using the ClusPro 3D modelling website and then the images edited using the iCn3D pdb file viewing website to highlight the interactions plus set the colours of each molecule being modelled. Details of how to do this are included in the Full Protocol on the Protocols page of this site.

HomerList2

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