de Mattos-Dutra A, Sampaio de Freitas M, Schröder N, Fogaça Lisboa C S, Pessoa-Pureur R, Wajner M
Departamento de Bioquímica, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
Exp Neurol. 1997 Oct;147(2):238-47. doi: 10.1006/exnr.1997.6602.
In the present study we demonstrate that propionic acid (PA), a metabolite that accumulates in large amounts in propionic acidemia, is able to decrease in vitro incorporation of [32P]ATP into neurofilament subunits (NF-M and NF-L) and alpha- and beta-tubulin. Considering that the endogenous phosphorylating system associated with the cytoskeletal fraction contains cAMP-dependent protein kinase (PKA), Ca2+/calmodulin protein kinase II (CaMKII), and protein phosphatase 1 (PP1), we first assayed the effect of the acid on the kinase activities by using the specific activators cAMP and Ca2+/calmodulin or the inhibitors PKAI or KN-93 for PKA and CaMKII, respectively. Results demonstrated that the acid totally inhibited the stimulatory effect of cAMP and interfered with the inhibitory effect of PKAI. In addition, PA partially prevented the stimulatory effect of Ca2+/calmodulin and interfered with the effect of KN-93. In addition, we demonstrated that PA totally inhibited in vitro dephosphorylation of neurofilament subunits and tubulins mediated by PP1 in brain slices pretreated with the acid. Taken together, these results demonstrate that PA inhibits the in vitro activities of PKA, CaMKII, and PP1 associated with the cytoskeletal fraction of the cerebral cortex of rats. This study suggests that PA at the same concentrations found in tissues from propionic acidemic children may alter phosphorylation of cytoskeletal proteins, which may contribute to the neurological dysfunction characteristic of propionic acidemia.
在本研究中，我们证明了丙酸（PA），一种在丙酸血症中大量积累的代谢产物，能够在体外降低[32P]ATP掺入神经丝亚基（NF-M和NF-L）以及α-和β-微管蛋白的量。鉴于与细胞骨架部分相关的内源性磷酸化系统包含cAMP依赖性蛋白激酶（PKA）、Ca2+/钙调蛋白蛋白激酶II（CaMKII）和蛋白磷酸酶1（PP1），我们首先分别使用特异性激活剂cAMP和Ca2+/钙调蛋白或PKA和CaMKII的抑制剂PKAI或KN-93来检测该酸对激酶活性的影响。结果表明，该酸完全抑制了cAMP的刺激作用，并干扰了PKAI的抑制作用。此外，PA部分阻止了Ca2+/钙调蛋白的刺激作用，并干扰了KN-93的作用。此外，我们证明了PA完全抑制了在用该酸预处理的脑片中由PP1介导的神经丝亚基和微管蛋白的体外去磷酸化。综上所述，这些结果表明PA抑制了与大鼠大脑皮质细胞骨架部分相关的PKA、CaMKII和PP1的体外活性。本研究表明，在丙酸血症儿童组织中发现的相同浓度的PA可能会改变细胞骨架蛋白的磷酸化，这可能导致丙酸血症特有的神经功能障碍。
