Based on the Multiple Sequence Alignment results generated by Clustal Omega (provided by Swiss Institute of Bioinformatics) and BOXSHADE 3.21, we can observe that the different isoforms of human procaspase-8 share a great deal of homology, as shown by the sections of the peptide that are highlighted in black. The fact that these residues are highly conserved across different isoforms indicate that they must have been essential in the proper functioning of the proteins.  Some of these residues correspond to functional domains, or important motifs for inter-protein interactions according to Pfam and Smart analysis (we have indicated the individual domains on the alignments). Therefore, comparing the differences in sequences of the isoforms can allow us to deduce how mutations (both deletions or insertions) or a change in the property/characteristic of certain amino acid residues might abolish or modulate the functions of a protein.

From the analysis of the abovementioned alignments, we can conclude:

1. Procaspase-8S contains only the first DED and a small part of the second DED, and lacks the entire C-terminal protease domain CASc. In vivo binding assays have suggested that this isoform of procaspase-8 can still bind to FADD during the formation of DICS. This provides evidence that the first DED in the pro-domain plays a crucial role in the initial binding of caspase-8 to FADD upon death stimuli [1]. To be more specific, it has been shown that the procaspase-8 utilizes the tyrosine residue in DED1 (Y8) to interact with the hydrophobic patch present on a2 or a5 helices of FADD [2].

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2. Apart from procaspase-8S, all the other isoforms share almost identical sequences for the two tandem DEDs, with procaspase-8a having a slight extension in the region between the two DED. Nevertheless, these isoforms all contain the highly conserved alpha helical bundle in their pro-domains, having 6 alpha helices present in each DED. The RxDL motif in a6 is highly conserved in all DED-containing proteins, and is suggested to interact with acidic residues glutamate or aspartate in a2. The resultant charged triad E/D-RxDL motif is characteristic of all DED family proteins, and is suggested to play a role in holding alpha helices 2 and 6 together, hence stabilising the entire DED globular structure [3].

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3. Procaspase-8L and caspase-8p41 both contain a truncated C-terminal domain. In the case of casp8p41, the lack of the catalytic C360, as well as the subsequent p10 subunit, makes it unable to dimerise and activate other normal procaspase-8. Thus, it stimulates cell death either by acting as a scaffold protein directing the recruitment of functional procaspase-8, or trigger apoptosis in caspase-8 independent manners [4]. In the case of procaspase-8L, there is an insertion of 136bp fragment which generates a stop codon, leading to the synthesis of pre-mature procaspase-8 with only a part of p18. Owing to the presence of intact DEDs, procasp-8L can still bind to FADD, but is unable to induce apoptosis. Therefore, it has the potential of inhibiting caspase-8 dependent apoptosis by competing with isoform 8a or b for the recruitment site of FADD.