Molecular regulatory mechanism of human myosin-7a.
Molecular regulatory mechanism of human myosin-7a.
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DOI：
10.1016/j.jbc.2023.105243
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发表时间：
2023-10
影响因子：
4.8
通讯作者：
Liu, Rong
中科院分区：
文献类型：
--
作者：
Hollo, Alexandra;Billington, Neil;Takagi, Yasuharu;Kengyel, Andras;Sellers, James R.;Liu, Rong
肌球蛋白-7a是一种基于肌动蛋白的运动蛋白，对视觉和听力至关重要。肌球蛋白-7a突变导致1型Usher综合征，这是人类最常见和最严重的聋盲形式。控制其机械化学的分子机制仍然知之甚少，主要是因为纯化稳定完整的蛋白质的困难。本研究在昆虫细胞中重组制备了完整的人肌球蛋白-7a全酶，并对其生化和运动特性进行了表征。与主要与钙调蛋白（CaM）相关的果蝇同源基因不同，我们发现人类肌球蛋白-7a利用一种独特的轻链组合，包括调节轻链、CaM和CaM样蛋白4。我们的研究结果进一步揭示了cam样蛋白4不作为Ca2+传感器，但在维持杠杆臂的结构功能完整性方面起着至关重要的作用。利用我们的重组蛋白系统，我们纯化了两个myosin-7a剪接异构体，这些异构体已被证明沿耳蜗张力异位轴差异表达。我们表明，尽管它们的N端只有11个氨基酸不同，但它们具有不同的机械酶性质。通过单分子体外运动测定，我们证明了人肌球蛋白-7a作为一种自抑制单体存在，当人工二聚化或与货物适应蛋白结合时，可以沿着肌动蛋白前进。这些结果表明肌球蛋白-7a可以在感觉系统中发挥多种作用，例如作为转运蛋白或锚定/力传感器。此外，我们的研究强调，人类肌球蛋白-7a已经进化出独特的调节元件，能够精确调节其机械特性，适合哺乳动物的听觉功能。
Myosin-7a is an actin-based motor protein essential for vision and hearing. Mutations of myosin-7a cause type 1 Usher syndrome, the most common and severe form of deafblindness in humans. The molecular mechanisms that govern its mechanochemistry remain poorly understood, primarily because of the difficulty of purifying stable intact protein. Here, we recombinantly produce the complete human myosin-7a holoenzyme in insect cells and characterize its biochemical and motile properties. Unlike the Drosophila ortholog that primarily associates with calmodulin (CaM), we found that human myosin-7a utilizes a unique combination of light chains including regulatory light chain, CaM, and CaM-like protein 4. Our results further reveal that CaM-like protein 4 does not function as a Ca2+ sensor but plays a crucial role in maintaining the lever arm’s structural–functional integrity. Using our recombinant protein system, we purified two myosin-7a splicing isoforms that have been shown to be differentially expressed along the cochlear tonotopic axis. We show that they possess distinct mechanoenzymatic properties despite differing by only 11 amino acids at their N termini. Using single-molecule in vitro motility assays, we demonstrate that human myosin-7a exists as an autoinhibited monomer and can move processively along actin when artificially dimerized or bound to cargo adaptor proteins. These results suggest that myosin-7a can serve multiple roles in sensory systems such as acting as a transporter or an anchor/force sensor. Furthermore, our research highlights that human myosin-7a has evolved unique regulatory elements that enable precise tuning of its mechanical properties suitable for mammalian auditory functions.
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DOI：
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发表时间：
2015-03
影响因子：
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作者：
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影响因子：
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影响因子：
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