Mutation of juxtamembrane cysteines in the tetraspanin CD81 affects palmitoylation and alters interaction with other proteins at the cell surface.

Palmitoylation of tetraspanins affects protein-protein interactions, suggesting a key role in the assembly of the tetraspanin web. Since palmitoylation occurs on intracellular cysteine residues, we examined whether mutating these residues in the human tetraspanin CD81 would affect the association of CD81 with other surface membrane proteins. Mutation of at least six of the eight juxtamembrane cysteines was required to completely eliminate detectable CD81 palmitoylation, indicating that several sites can be palmitoylated. Interestingly, these mutated proteins exhibited reduced cell surface detection by antibody compared to wild-type CD81, but this was not due to differences in the level of protein expression, trafficking to the cell surface, protein stability, or anti-CD81 antibody binding affinity. Instead, the mutant CD81 proteins appeared to be partially hidden from detection by anti-CD81 antibody, presumably due to altered interactions with other proteins at the cell surface. Associations with the known CD81-interacting proteins CD9 and EWI-2 were also impaired with the mutant CD81 proteins. Taken together, these findings indicate that mutation of juxtamembrane cysteines alters the interaction of CD81 with other proteins, either because of reduced palmitoylation, structural alterations in the mutant proteins, or a combination of both factors, and this affects the CD81 microenvironment on the cell surface.

Identification and functional characterization of AMVp33, a novel homolog of the baculovirus caspase inhibitor p35 found in Amsacta moorei entomopoxvirus.

Members of the baculovirus p35 gene family encode proteins that specifically inhibit caspases, cysteine proteases that are involved in apoptosis. To date, p35 homologs have only been found in baculoviruses. We have identified AMVp33, a gene from Amsacta moorei entomopoxvirus with low but significant homology to baculovirus p35 genes. Expression of AMVp33 blocked apoptosis in several different insect and human cell lines. Purified recombinant P33 protein was an efficient inhibitor of insect and human effector caspases, but not initiator caspases. P33 was cleaved by effector caspases, and the resulting cleavage fragments stably associated with the caspases. Mutation of the predicted caspase cleavage site in P33 eliminated cleavage, caspase inhibition and anti-apoptotic function. Thus, AMVp33 encodes a caspase inhibitor similar to baculovirus P35 with a preference for effector caspases. This is the first report of a p35 homolog from any viral or cellular genome outside of the baculovirus family.

The baculovirus anti-apoptotic protein Op-IAP does not inhibit Drosophila caspases or apoptosis in Drosophila S2 cells and instead sensitizes S2 cells to virus-induced apoptosis.

The Op-IAP protein from the baculovirus Orgyia pseudotsugata M nucleopolyhedrovirus (OpMNPV) is highly effective at inhibiting apoptosis triggered by a variety of different stimuli in lepidopteran cells as well as in several different mammalian cell types, suggesting that it functions at a highly conserved step in the apoptotic pathway. However, the mechanism by which Op-IAP inhibits apoptosis is unclear. Since some IAP proteins can bind and inhibit caspases, we tested whether Op-IAP could inhibit the activity of caspases from Drosophila melanogaster. We found that recombinant Op-IAP protein was not able to bind or directly inhibit the activity of the Drosophila caspases DRONC, DrICE, or DCP-1 in vitro. In addition, expression of Op-IAP was unable to inhibit apoptosis triggered by either actinomycin D or UV light in D. melanogaster S2 cells. Surprisingly, Op-IAP expression in S2 cells enhanced apoptosis caused by baculovirus infection, but did not cause increased sensitivity to either actinomycin D or UV damage-induced apoptosis. The observation that Op-IAP cannot inhibit these insect caspases suggests that it functions by a mechanism that does not involve direct caspase inhibition.