An aliquot of total cell pro tein was retained and the remaining cells were res

The interplay between S nitrosylation and H2O2 type oxidation of BACE1 at the molecular level is not yet clear, albeit both occur at certain critical Cys residues. Based on our finding that NO supresses BACE1, we speculate that S nitrosylation and H2O2 type oxidation occur on overlap ping Cys residues on BACE1, nitrosylation precludes ABT-737 価格 further oxidation for enzymatic activation and thus represents a self defensive or house keeping mechanism. Among the 11 Cys residues on BACE1, it is known that six Cys residues form three pairs of intramolecular dis ulfide bonds in mature BACE1 which is essential for its membrane association and protein maturation but is not required for its enzymatic activity.

Since the pattern of BACE1 S nitrosylation shows that it occurs predominantly on the mature form of BACE1, we reason that the three pairs of Cys in the enzy matic pocket are not likely sites for nitrosylation. Indeed, our preliminary study used site directed muta genesis AEB071 構造 to analyze individual BACE1 mutants with each Cys mutated to Ala and showed that Cys mutation at the positions 216, 278, 330, 380, 420, 443, or 466 resulted in lack of mature BACE1 proteins. Substitution of each of the four Cys residues in the cytoplasmic tails, which are also the S palmitoylation sites, had different results, the C483A mutant abolished mature BACE1, making it dif ficult to assess its contribution as a nitrosylation site, Cys478A and Cys482A mutants appeared to show reduced BACE1 nitrosylation and likely represent S nitrosylation sites under physiological conditions.

It should be pointed out that the results of this type of semi quantitative analysis are not sufficient to determine the molecular basis of nitrosylation sites unambiguously. In particular, the cytoplasmic tail has been reported to be non essential for the enzymatic activity of BACE1, it remains unclear how the nitrosylation AG-014699 溶解度 affects BACE1 activity. Additional research to determine the molecular basis of the nitrosylation and oxidation events by quantitative mass spectrometry, as well as further analysis of BACE1 and subcellular trafficking, localiza tion and distribution in lipid rafts using cell biology approaches, are necessary to clarify the mechanism. Conclusion In summary, our studies have demonstrated that NO exerts differential regulation on BACE1 at low and modest to high concentrations, suppressing BACE1 transcriptionally or post translationally through S nitrosy lation, respectively.

S nitrosylation may represent a basic regulatory mechanism for maintaining BACE1 at physiolo gical levels, outside of events that challenge the brain with a wave of high oxidative stressors which upregulates and activates BACE1. As BACE1 represents a favored thera peutic target for developing anti AD agents, pharmacolo gical inhibitors of BACE1 have been actively pursued for more than a decade. Despite significant progress, the development of specific cell permeable drugs that pene trate into the brain remains a challenge. Due to the recent discouraging results from Eli Lillys trials on a g secretase inhibitor showing worsening cognitive performance in AD patients, efforts to discover a novel mechanism to modulate secretases is of particular importance.