Identification of Filamin A Mechanobinding Partner II: Fimbacin Is a Novel Actin Cross-Linking and Filamin A Binding Protein
Identification of Filamin A Mechanobinding Partner II: Fimbacin Is a Novel Actin Cross-Linking and Filamin A Binding Protein
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细丝蛋白 A 机械结合伙伴 II 的鉴定：Fimbacin 是一种新型肌动蛋白交联和细丝蛋白 A 结合蛋白
DOI：
10.1021/acs.biochem.9b00101
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发表时间：
2019
期刊：
影响因子：
2.9
通讯作者：
Fumihiko Nakamura
中科院分区：
文献类型：
--
作者：
Wang J.;Fumihiko Nakamura
细丝蛋白A（FLNA）是一种肌动蛋白交联蛋白，通过暴露重复序列21（R21）上的隐蔽结合位点与整联蛋白相互作用而充当机械传感器和机械转换器。在这里，我们研究了是否有任何其他生物分子与隐蔽结合位点相互作用。使用蛋白质组学和anin silicoscreating的FLNA结合基序，我们确定和表征的蛋白质称为fimbacin（细丝蛋白mechanobinding肌动蛋白交联蛋白），编码在LUZP 1基因，作为一种新的FLNA结合伴侣。Fimbacin不与典型的全长FLNA相互作用，但FLNA R21的隐蔽整合素结合位点的暴露使得Fimbacin能够相互作用。我们已经确定了两个FLNA结合位点fimbacin和确定的相互作用的关键氨基酸残基。我们还发现fimbacin本身是一种新的肌动蛋白交联蛋白，并定位了肌动蛋白结合位点的氨基酸残基400-500。Fimbacin通过氨基末端结构域寡聚化（在尺寸排阻色谱上估计为八聚体），所述氨基末端结构域被预测为卷曲螺旋以交联肌动蛋白丝。当表达时，fimbacin定位于组织培养细胞中的肌动蛋白应力纤维。虽然与FLNA的相互作用是没有必要的fimbacin与F-肌动蛋白共定位，荧光恢复后的光漂白（FRAP）显示，他们的相互作用稳定fimbacin的肌动蛋白细胞骨架和抑制Rho-激酶，肌球蛋白II的上游激活剂，也降低了相互作用，大概是由于损失的内部机械应力。总之，这些数据确定fimbacin作为一种新的肌动蛋白交联蛋白，与FLNA mechanosensing结构域R21相互作用。
Filamin A (FLNA), an actin cross-linking protein, acts as a mechanosensor and mechanotransducer by exposing the cryptic binding site on repeat 21 (R21) to interact with integrin. Here, we investigated if any other biological molecule interacts with the cryptic binding site. Using proteomics and anin silicoscreening for a FLNA-binding motif, we identified and characterized a protein termed fimbacin (filamin mechanobinding actin cross-linking protein), encoded in the LUZP1 gene, as a novel FLNA-binding partner. Fimbacin does not interact with canonical full-length FLNA, but the exposure of a cryptic integrin-binding site of FLNA R21 enables fimbacin to interact. We have identified two FLNA binding sites on fimbacin and determined critical amino acid residues for the interaction. We also found that fimbacin itself is a new actin cross-linking protein and mapped the actin-binding site on amino acid residues 400–500. Fimbacin oligomerizes (estimated as an octamer on size exclusion chromatography) through the amino-terminal domain that is predicted to be a coiled-coil to cross-link actin filaments. When expressed, fimbacin localized to actin stress fibers in tissue culture cells. Although the interaction with FLNA is not necessary for fimbacin to colocalize with F-actin, fluorescent recovery after photobleaching (FRAP) revealed that their interaction stabilizes fimbacin on the actin cytoskeleton and that inhibition of Rho-kinase, an upstream activator of myosin II, also decreases the interaction presumably due to a loss of internal mechanical stress. Taken together, these data identify fimbacin as a new actin cross-linking protein that interacts with the FLNA mechanosensing domain R21.