Department of Experimental and Clinical Biomedical Sciences "Mario Serio", University of Florence, 50134, Florence, Italy.
Department of Chemistry "Ugo Schiff", University of Florence, 50019, Florence, Italy.
Cell Mol Biol Lett. 2024 Jul 13;29(1):104. doi: 10.1186/s11658-024-00615-4.
Frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-TDP), amyotrophic lateral sclerosis (ALS) and limbic-predominant age-related TDP-43 encephalopathy (LATE) are associated with deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons. One complexity of this process lies in the ability of TDP-43 to form liquid-phase membraneless organelles in cells. Previous work has shown that the recombinant, purified, prion-like domain (PrLD) forms liquid droplets in vitro, but the behaviour of the complementary fragment is uncertain.
We have purified such a construct without the PrLD (PrLD-less TDP-43) and have induced its phase separation using a solution-jump method and an array of biophysical techniques to study the morphology, state of matter and structure of the TDP-43 assemblies.
The fluorescent TMR-labelled protein construct, imaged using confocal fluorescence, formed rapidly (< 1 min) round, homogeneous and 0.5-1.0 µm wide assemblies which then coalesced into larger, yet round, species. When labelled with AlexaFluor488, they initially exhibited fluorescence recovery after photobleaching (FRAP), showing a liquid behaviour distinct from full-length TDP-43 and similar to PrLD. The protein molecules did not undergo major structural changes, as determined with circular dichroism and intrinsic fluorescence spectroscopies. This process had a pH and salt dependence distinct from those of full-length TDP-43 and its PrLD, which can be rationalized on the grounds of electrostatic forces.
Similarly to PrLD, PrLD-less TDP-43 forms liquid droplets in vitro through liquid-liquid phase separation (LLPS), unlike the full-length protein that rather undergoes liquid-solid phase separation (LSPS). These results offer a rationale of the complex electrostatic forces governing phase separation of full-length TDP-43 and its fragments. On the one hand, PrLD-less TDP-43 has a low pI and oppositively charged domains, and LLPS is inhibited by salts, which attenuate inter-domain electrostatic attractions. On the other hand, PrLD is positively charged due to a high isoionic point (pI) and LLPS is therefore promoted by salts and pH increases as they both reduce electrostatic repulsions. By contrast, full-length TDP-43 undergoes LSPS most favourably at its pI, with positive and negative salt dependences at lower and higher pH, respectively, depending on whether repulsive or attractive forces dominate, respectively.
具有泛素阳性包涵体的额颞叶变性（FTLD-TDP）、肌萎缩侧索硬化症（ALS）和边缘为主的与年龄相关的 TDP-43 脑病（LATE）与神经元中 TAR DNA 结合蛋白 43（TDP-43）的细胞质包涵体沉积有关。这个过程的一个复杂性在于 TDP-43 在细胞中形成无膜液滴的能力。以前的工作表明，重组的、纯化的、类朊病毒结构域（PrLD）在体外形成液滴，但互补片段的行为不确定。
我们已经纯化了没有 PrLD 的这种构建体（无 PrLD 的 TDP-43），并使用溶液跃变方法和一系列生物物理技术诱导其相分离，以研究 TDP-43 组装体的形态、物相和结构。
用共聚焦荧光成像的荧光标记 TMR 蛋白构建体迅速（<1 分钟）形成圆形、均匀的 0.5-1.0 µm 宽的组装体，然后融合成更大的但仍是圆形的物种。当用 AlexaFluor488 标记时，它们最初表现出光漂白后荧光恢复（FRAP），表现出与全长 TDP-43 不同的液体行为，类似于 PrLD。圆二色性和内源荧光光谱学表明，蛋白质分子没有发生重大结构变化。这个过程的 pH 值和盐依赖性与全长 TDP-43 和其 PrLD 的不同，可以根据静电作用力来解释。
与全长 TDP-43 不同，无 PrLD 的 TDP-43 通过液-液相分离（LLPS）在体外形成液滴，而不是经历液-固相分离（LSPS）。这些结果为全长 TDP-43 及其片段的相分离提供了复杂静电作用力的基本原理。一方面，无 PrLD 的 TDP-43 的等电点较低且带有带正电荷的结构域，盐会抑制 LLPS，从而减弱结构域之间的静电吸引力。另一方面，由于高等电点（pI），PrLD 带正电荷，因此盐的存在和 pH 值的增加都会促进 LLPS，因为它们分别降低了静电排斥力。相比之下，全长 TDP-43 在其等电点下最有利于进行 LSPS，在较低和较高 pH 值下分别具有正盐依赖性和负盐依赖性，这取决于排斥力还是吸引力占主导地位。
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