Honarmand Ebrahimi Kourosh, Dienemann Christian, Hoefgen Sandra, Than Manuel E, Hagedoorn Peter-Leon, Hagen Wilfred R
Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
PLoS One. 2013 Aug 19;8(8):e72177. doi: 10.1371/journal.pone.0072177. eCollection 2013.
The ubiquitous 24-meric iron-storage protein ferritin and multicopper oxidases such as ceruloplasmin or hephaestin catalyze oxidation of Fe(II) to Fe(III), using molecular oxygen as oxidant. The ferroxidase activity of these proteins is essential for cellular iron homeostasis. It has been reported that the amyloid precursor protein (APP) also has ferroxidase activity. The activity is assigned to a ferroxidase site in the E2 domain of APP. A synthetic 22-residue peptide that carries the putative ferroxidase site of E2 domain (FD1 peptide) has been claimed to encompass the same activity. We previously tested the ferroxidase activity of the synthetic FD1 peptide but we did not observe any activity above the background oxidation of Fe(II) by molecular oxygen. Here we used isothermal titration calorimetry to study Zn(II) and Fe(II) binding to the natural E2 domain of APP, and we employed the transferrin assay and oxygen consumption measurements to test the ferroxidase activity of the E2 domain. We found that this domain neither in the presence nor in the absence of the E1 domain binds Fe(II) and it is not able to catalyze the oxidation of Fe(II). Binding of Cu(II) to the E2 domain did not induce ferroxidase activity contrary to the presence of redox active Cu(II) centers in ceruloplasmin or hephaestin. Thus, we conclude that E2 or E1 domains of APP do not have ferroxidase activity and that the potential involvement of APP as a ferroxidase in the pathology of Alzheimer's disease must be re-evaluated.
普遍存在的24聚体铁储存蛋白铁蛋白以及多铜氧化酶（如铜蓝蛋白或铁转运蛋白）以分子氧作为氧化剂，催化Fe(II)氧化为Fe(III)。这些蛋白质的铁氧化酶活性对于细胞铁稳态至关重要。据报道，淀粉样前体蛋白（APP）也具有铁氧化酶活性。该活性归因于APP的E2结构域中的一个铁氧化酶位点。一种携带E2结构域假定铁氧化酶位点的合成22肽（FD1肽）据称具有相同的活性。我们之前测试了合成的FD1肽的铁氧化酶活性，但未观察到任何高于分子氧对Fe(II)背景氧化的活性。在这里，我们使用等温滴定量热法研究Zn(II)和Fe(II)与APP天然E2结构域的结合，并采用转铁蛋白测定法和氧消耗测量来测试E2结构域的铁氧化酶活性。我们发现，无论是否存在E1结构域，该结构域都不结合Fe(II)，并且无法催化Fe(II)的氧化。与铜蓝蛋白或铁转运蛋白中存在氧化还原活性Cu(II)中心相反，Cu(II)与E2结构域的结合并未诱导铁氧化酶活性。因此，我们得出结论，APP的E2或E1结构域不具有铁氧化酶活性，并且APP作为铁氧化酶在阿尔茨海默病病理学中的潜在作用必须重新评估。
