Greve Johannes N, Schwäbe Frederic V, Pokrant Thomas, Faix Jan, Di Donato Nataliya, Taft Manuel H, Manstein Dietmar J
Institute for Biophysical Chemistry and Structural Biochemistry, Medizinische Hochschule Hannover, Hannover 30625, Germany.
Institute for Clinical Genetics, University Hospital, TU Dresden, Dresden 01307, Germany.
Eur J Cell Biol. 2022 Apr;101(2):151216. doi: 10.1016/j.ejcb.2022.151216. Epub 2022 Mar 15.
Heterozygous dominant mutations in the ubiquitously produced cytoskeletal β-actin isoform lead to a broad range of human disease phenotypes, which are currently classified as three distinct clinical entities termed Baraitser-Winter-Cerebrofrontofacial syndrome (BWCFF), ACTB-associated pleiotropic malformation syndrome with intellectual disability (ACTB-PMSID), and ACTB-associated syndromic thrombocytopenia (ACTB-AST). The latter two are distinguishable from BWCFF by the presence of milder craniofacial features and less pronounced developmental abnormalities, or the absence of craniofacial features in combination with a characteristic thrombocytopenia with platelet anisotropy. Production and correct function of β-actin is required for multiple essential processes in all types of cells. Directed cell migration, cytokinesis and morphogenesis are amongst the functions that are supported by β-actin. Here we report the recombinant production and biochemical characterization of the ACTB-AST mutant p.S368fs, resulting in an altered sequence in the C-terminal region of β-actin that includes a replacement of the last 8 residues and an elongation of the molecule by 4 residues. The mutation affects a region important for actin polymerization and actin-profilin interaction. Accordingly, we measured markedly reduced rates of nucleation and polymerization during spontaneous actin assembly and lower affinity of p.S368fs for human profilin-1. The reduced affinity is also reflected in the lower propensity of profilin-1 to extend the nucleation phase of p.S368fs. While localized in close proximity to actin-cofilin and actin-myosin interfaces, we determined only minor effects of the mutation on the interaction of mutant filaments with cofilin and myosin family members. However, allosteric effects on sites distant from the mutation manifest themselves in a 7.9 °C reduction in thermal denaturation temperature, a 2-fold increase in the observed IC for DNase-I, and changes in nucleotide exchange kinetics. Our results support a disease mechanism involving impaired actin dynamics and function through disruption of actin-profilin interactions and further exacerbated by allosteric perturbations.
普遍产生的细胞骨架β-肌动蛋白异构体中的杂合显性突变会导致一系列广泛的人类疾病表型，目前这些表型被归类为三种不同的临床实体，即巴拉伊泽-温特-脑额面部综合征（BWCFF）、与智力残疾相关的ACTB多效性畸形综合征（ACTB-PMSID）以及与ACTB相关的综合征性血小板减少症（ACTB-AST）。后两者与BWCFF的区别在于，它们具有较轻微的颅面特征和不太明显的发育异常，或者没有颅面特征，同时伴有特征性的血小板减少和血小板大小不均。β-肌动蛋白的产生和正确功能是所有类型细胞中多种基本过程所必需的。定向细胞迁移、胞质分裂和形态发生等功能都由β-肌动蛋白支持。在此，我们报告了ACTB-AST突变体p.S368fs的重组生产和生化特性，该突变导致β-肌动蛋白C末端区域的序列改变，包括最后8个残基的替换以及分子延长4个残基。该突变影响了对肌动蛋白聚合和肌动蛋白-肌动蛋白结合蛋白相互作用至关重要的区域。因此，我们测量到在自发肌动蛋白组装过程中，成核和聚合速率显著降低，并且p.S368fs与人肌动蛋白结合蛋白-1的亲和力较低。亲和力降低还反映在肌动蛋白结合蛋白-1延长p.S368fs成核阶段的倾向较低。虽然该突变位于靠近肌动蛋白-丝切蛋白和肌动蛋白-肌球蛋白界面的位置，但我们确定该突变对突变细丝与丝切蛋白和肌球蛋白家族成员相互作用的影响较小。然而，对远离突变位点的变构效应表现为热变性温度降低7.9°C，DNase-I的观察到的IC增加2倍，以及核苷酸交换动力学的变化。我们的结果支持一种疾病机制，即通过破坏肌动蛋白-肌动蛋白结合蛋白相互作用导致肌动蛋白动力学和功能受损，并因变构扰动而进一步加剧。
