Hillier James, Allcott Gemma J, Guest Laura A, Heaselgrave Wayne, Tonks Alex, Conway Myra E, Cherry Amy L, Coles Steven J
School of Science & the Environment, University of Worcester, Worcester WR2 6AJ, UK.
College of Life & Health Sciences, School of Biosciences, Aston University, Birmingham B4 7ET, UK.
Antioxidants (Basel). 2022 Mar 31;11(4):683. doi: 10.3390/antiox11040683.
The cytosolic branched-chain aminotransferase (BCAT1) has received attention for its role in myeloid leukaemia development, where studies indicate metabolic adaptations due to BCAT1 up-regulation. BCAT1, like the mitochondria isoform (BCAT2), shares a conserved CXXC motif ~10 Å from the active site. This CXXC motif has been shown to act as a 'redox-switch' in the enzymatic regulation of the BCAT proteins, however the response to reactive oxygen species (ROS) differs between BCAT isoforms. Studies indicate that the BCAT1 CXXC motif is several orders of magnitude less sensitive to the effects of ROS compared with BCAT2. Moreover, estimation of the reduction mid-point potential of BCAT1, indicates that BCAT1 is more reductive in nature and may possess antioxidant properties. Therefore, the aim of this study was to further characterise the BCAT1 CXXC motif and evaluate its role in acute myeloid leukaemia. Our biochemical analyses show that purified wild-type (WT) BCAT1 protein could metabolise HO in vitro, whereas CXXC motif mutant or WT BCAT2 could not, demonstrating for the first time a novel antioxidant role for the BCAT1 CXXC motif. Transformed U937 AML cells over-expressing WT BCAT1, showed lower levels of intracellular ROS compared with cells over-expressing the CXXC motif mutant (CXXS) or Vector Controls, indicating that the BCAT1 CXXC motif may buffer intracellular ROS, impacting on cell proliferation. U937 AML cells over-expressing WT BCAT1 displayed less cellular differentiation, as observed by a reduction of the myeloid markers; CD11b, CD14, CD68, and CD36. This finding suggests a role for the BCAT1 CXXC motif in cell development, which is an important pathological feature of myeloid leukaemia, a disease characterised by a block in myeloid differentiation. Furthermore, WT BCAT1 cells were more resistant to apoptosis compared with CXXS BCAT1 cells, an important observation given the role of ROS in apoptotic signalling and myeloid leukaemia development. Since CD36 has been shown to be Nrf2 regulated, we investigated the expression of the Nrf2 regulated gene, . Our data show that the expression of was downregulated in transformed U937 AML cells overexpressing WT BCAT1, which taken with the reduction in CD36 implicates less Nrf2 activation. Therefore, this finding may implicate the BCAT1 CXXC motif in wider cellular redox-mediated processes. Altogether, this study provides the first evidence to suggest that the BCAT1 CXXC motif may contribute to the buffering of ROS levels inside AML cells, which may impact ROS-mediated processes in the development of myeloid leukaemia.
胞质支链氨基转移酶（BCAT1）因其在髓系白血病发展中的作用而受到关注，研究表明BCAT1上调会导致代谢适应。BCAT1与线粒体异构体（BCAT2）一样，在距活性位点约10 Å处有一个保守的CXXC基序。该CXXC基序已被证明在BCAT蛋白的酶促调节中充当“氧化还原开关”，然而BCAT异构体对活性氧（ROS）的反应有所不同。研究表明，与BCAT2相比，BCAT1的CXXC基序对ROS作用的敏感性要低几个数量级。此外，对BCAT1还原中点电位的估计表明，BCAT1本质上更具还原性，可能具有抗氧化特性。因此，本研究的目的是进一步表征BCAT1的CXXC基序，并评估其在急性髓系白血病中的作用。我们的生化分析表明，纯化的野生型（WT）BCAT1蛋白在体外可以代谢HO，而CXXC基序突变体或WT BCAT2则不能，首次证明了BCAT1的CXXC基序具有新的抗氧化作用。与过表达CXXC基序突变体（CXXS）或载体对照的细胞相比，过表达WT BCAT1的转化U937 AML细胞显示出较低水平的细胞内ROS，这表明BCAT1的CXXC基序可能缓冲细胞内ROS，影响细胞增殖。过表达WT BCAT1的U937 AML细胞显示出较少的细胞分化，通过髓系标志物CD11b、CD14、CD68和CD36的减少可以观察到。这一发现表明BCAT1的CXXC基序在细胞发育中起作用，这是髓系白血病的一个重要病理特征，髓系白血病是一种以髓系分化受阻为特征的疾病。此外，与CXXS BCAT1细胞相比，WT BCAT1细胞对凋亡更具抗性，鉴于ROS在凋亡信号传导和髓系白血病发展中的作用，这是一个重要的观察结果。由于已证明CD36受Nrf2调节，我们研究了Nrf2调节基因的表达。我们的数据表明，在过表达WT BCAT1的转化U937 AML细胞中，该基因的表达下调，这与CD36的减少一起表明Nrf2激活较少。因此，这一发现可能意味着BCAT1的CXXC基序参与了更广泛的细胞氧化还原介导的过程。总之，本研究提供了首个证据表明BCAT1的CXXC基序可能有助于缓冲AML细胞内的ROS水平，这可能影响髓系白血病发展中ROS介导的过程。
