A more definitive conclusion has to await high resolution structural determination by X ray crystallog raphy. R96L, a missense mutation found in patients with ALS also appears to be a gain of function mutation with an enhanced binding with Rab8 and TfR. The binding was nevertheless somewhat weaker than that seen with E50K. The weaker binding may Tipifarnib buy be the basis why the R96L phenotypes were milder than those of E50K. L157A mutation has not been identified clinically in any patients to date and is more than likely not disease causing or related. As stated above, the mutation may lead to obliteration of the leucine zipper in the opti neurin sequence. L157A mutant has been shown to interact with Rab8 and TfR in a much reduced capacity, suggesting that the optineurin associ ation with Rab8 and TfR requires, at least in part, an in tact leucine zipper motif.
No perinuclear foci were observed with L157A or with two mutations in the UBD domain, D474N and E478G. It seems that the foci formation was abrogated by muta tions in either the leucine zipper or the UBD domain. Foci were also not noted when cells were transfected to express optineurin mutations fragments with sequence deletion in either Inhibitors,Modulators,Libraries or both of the domains such as 1 209, Q398X, 1 424, and 217 577. The intact sequences of both leucine zipper and UBD domains Inhibitors,Modulators,Libraries are thus concluded to be required for the perinuclear foci formation phenotype. This notion is somewhat differ ent Inhibitors,Modulators,Libraries from that described in a recent report in which aggregates were seen in yeast with fragments 1 251, 1 398, 182 577, and 98 398, as long as one of the coiled coil domains was present.
The disparity in results could be related to difference in the systems used, Inhibitors,Modulators,Libraries and also the definition Inhibitors,Modulators,Libraries or pattern of foci or aggregates formed. While all optineurin phenotypes are observed when foci are formed with mutants, the formation does not necessarily predict the other phe notypes. One example is the fragment Q398X, which, with no foci manifestation, resulted in Golgi frag mentation, defective transferrin uptake and apoptosis. The foci are likely formed by self binding of optineurin mole cules as well as their interactions with proteins including Rab8, myosin VI and TfR, requiring at least in part, intact leucine zipper and UBD motifs. Overabundance or accu mulation of the protein fragment and perturbation of the protein interactions conceivably would drive foci forma tion.
The role or significance of the foci observed is at present uncertain. Optineurin foci are reminiscent of the inclusion bodies, Lewy bodies or aggresomes detected in most neurodegenerative diseases. The inclu sion bodies and aggresomes once were considered to be the culprit for neurodegenerative diseases. More recent evidence however suggests that they may play protective role by sequestering toxic, misfolded protein species and providing the cells with an opportunity of delayed protein degradation. They may also inactivate the proteasome and mediate cytotoxicity.