caspases are well known for excutionars of apoptosis but caspases are involved in even more important functions like cell differentiation and proliferation but i think unlike apoptosis where cell is undergoing demise, caspases activity maybe constrained during cell differentiation or activation kinetics may be different for both these process ,can any one suggest me how to find the targets of caspases in cells under going differentiation and how to check what are the intaracting patnars which are regulating caspases.
I guess you could go three ways.
- Coimmunoprecipitate the caspase and have the product analysed by MS
- Use bioinformatics. You could downdoad the annotated protein sequence and create a database that you could analyse by the consensus sequence of the caspase you are interested. Afterwards you could screen the possible targets for caspase colocalization (cytoplasm, nucleus, etc). Finally you would need validate your data. You could transfect both caspase and the proteins of interest and access cleavage by western blot.
- use caspase inhibitor and have a whole proteomic analysis and check the difference between non inhibit and inhibit cells.
Hi,
There is additional possibility of bioinformatics prediction of potential caspase targets. CutDB database (http://cutdb.burnham.org) contains list of proteins hydrolyzed by caspases. Database contains also short fragments containing four amino acid residues preceding and four following cleaved bond. You may screen major protein database (e.g. UniProt) using protein sequences as a queries and you can find homologs of these proteins. You can also use short fragments as a queries. You can find set of proteins containing identical fragment. I tried fwe minutes ago with the sequence ALADSLGK (proteolytic event No 3375 in CutDB). I used NCBI BLAST available at the website of European Bioinformatics Institute, with matrix PAM 30, expected threshold 1000 and sort according to high score. I found 96 proteins containing this fragment.
Reference:
https://www.researchgate.net/publication/269103857_The_Occurrence_of_Sequences_Identical_with_Epitopes_from_the_Allergen_Pen_a_10102_Among_Food_and_Non-Food_Proteins
Article The Occurrence of Sequences Identical with Epitopes from the...
I agree 100% with the previous two answers. Bioinformatics tools would be a great starting point and for generating experimental evidence, inhibitors/inducers based studies would be critical. Novus' poster on apoptosis has outlined various signaling proteins including caspases as well as their inhibitors which are highly useful for mechanistic studies in apoptotic cell death pathway: http://images.novusbio.com/design/apoptosis_poster.pdf
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