Reyes Juan Sebastián, Fuentes-Lemus Eduardo, Aspée Alexis, Davies Michael J, Monasterio Octavio, López-Alarcón Camilo
Departamento de Química Física, Facultad de Química y de Farmacia, Pontificia Universidad Católica de Chile, Chile; Laboratorio de Biología Estructural y Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Chile.
Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Denmark.
Free Radic Biol Med. 2021 Apr;166:53-66. doi: 10.1016/j.freeradbiomed.2021.02.003. Epub 2021 Feb 13.
Oxidation and inactivation of FtsZ is of interest due to the key role of this protein in bacterial cell division. In the present work, we studied peroxyl radical (from AAPH, 2,2'-azobis(2-methylpropionamidine)dihydrochloride) mediated oxidation of the highly stable FtsZ protein (MjFtsZ) from M. jannaschii, a thermophilic microorganism. MjFtsZ contains eleven Met, and single Tyr and Trp residues which would be expected to be susceptible to oxidation. We hypothesized that exposure of MjFtsZ to AAPH-derived radicals would induce Met oxidation, and cross-linking (via di-Tyr and di-Trp formation), with concomitant loss of its functional polymerization and depolymerization (GTPase) activities. Solutions containing MjFtsZ and AAPH (10 or 100 mM) were incubated at 37 °C for 3 h. Polymerization/depolymerization were assessed by light scattering, while changes in mass were analyzed by SDS-PAGE. Amino acid consumption was quantified by HPLC with fluorescence detection, or direct fluorescence (Trp). Oxidation products and modifications at individual Met residues were quantified by UPLC with mass detection. Oxidation inhibited polymerization-depolymerization activity, and yielded low levels of irreversible protein dimers. With 10 mM AAPH only Trp and Met were consumed giving di-alcohols, kynurenine and di-Trp (from Trp) and the sulfoxide (from Met). With 100 mM AAPH low levels of Tyr oxidation (but not di-Tyr formation) were also observed. Correlation with the functional analyses indicates that Met oxidation, and particularly Met164 is the key driver of MjFtsZ inactivation, probably as a result of the position of this residue at the protein-protein interface of longitudinal interactions and in close proximity to the GTP binding site.
由于FtsZ蛋白在细菌细胞分裂中起关键作用，其氧化和失活备受关注。在本研究中，我们研究了过氧自由基（来自2,2'-偶氮二异丁脒二盐酸盐（AAPH））介导的嗜热微生物詹氏甲烷球菌中高度稳定的FtsZ蛋白（MjFtsZ）的氧化。MjFtsZ含有11个甲硫氨酸残基，以及单个酪氨酸和色氨酸残基，预计这些残基易被氧化。我们推测，MjFtsZ暴露于AAPH衍生的自由基会诱导甲硫氨酸氧化和交联（通过二酪氨酸和二色氨酸形成），同时其功能性聚合和解聚（GTP酶）活性丧失。将含有MjFtsZ和AAPH（10或100 mM）的溶液在37℃孵育3小时。通过光散射评估聚合/解聚，同时通过SDS-PAGE分析质量变化。通过具有荧光检测的HPLC或直接荧光（色氨酸）对氨基酸消耗进行定量。通过具有质量检测的UPLC对单个甲硫氨酸残基处的氧化产物和修饰进行定量。氧化抑制了聚合-解聚活性，并产生了低水平的不可逆蛋白质二聚体。使用10 mM AAPH时，仅色氨酸和甲硫氨酸被消耗，生成二醇、犬尿氨酸和二色氨酸（来自色氨酸）以及亚砜（来自甲硫氨酸）。使用100 mM AAPH时，还观察到低水平的酪氨酸氧化（但未形成二酪氨酸）。与功能分析的相关性表明，甲硫氨酸氧化，尤其是甲硫氨酸164是MjFtsZ失活的关键驱动因素，这可能是由于该残基在纵向相互作用的蛋白质-蛋白质界面处的位置以及靠近GTP结合位点的缘故。
