Physics Department, Syracuse University, New York, NY 13244, USA.
Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221, USA.
Biomolecules. 2023 Mar 30;13(4):620. doi: 10.3390/biom13040620.
In cells, microtubule location, length, and dynamics are regulated by a host of microtubule-associated proteins and enzymes that read where to bind and act based on the microtubule "tubulin code," which is predominantly encoded in the tubulin carboxy-terminal tail (CTT). Katanin is a highly conserved AAA ATPase enzyme that binds to the tubulin CTTs to remove dimers and sever microtubules. We have previously demonstrated that short CTT peptides are able to inhibit katanin severing. Here, we examine the effects of CTT sequences on this inhibition activity. Specifically, we examine CTT sequences found in nature, alpha1A (TUBA1A), detyrosinated alpha1A, Δ2 alpha1A, beta5 (TUBB/TUBB5), beta2a (TUBB2A), beta3 (TUBB3), and beta4b (TUBB4b). We find that these natural CTTs have distinct abilities to inhibit, most noticeably beta3 CTT cannot inhibit katanin. Two non-native CTT tail constructs are also unable to inhibit, despite having 94% sequence identity with alpha1 or beta5 sequences. Surprisingly, we demonstrate that poly-E and poly-D peptides are capable of inhibiting katanin significantly. An analysis of the hydrophobicity of the CTT constructs indicates that more hydrophobic polypeptides are less inhibitory than more polar polypeptides. These experiments not only demonstrate inhibition, but also likely interaction and targeting of katanin to these various CTTs when they are part of a polymerized microtubule filament.
在细胞中，微管的位置、长度和动态受到许多微管相关蛋白和酶的调节，这些蛋白和酶根据微管的“微管蛋白密码”读取结合的位置并发挥作用，而微管蛋白密码主要编码在微管羧基末端尾巴（CTT）上。Katanin 是一种高度保守的 AAA ATP 酶，它与微管 CTT 结合以去除二聚体并切断微管。我们之前已经证明，短的 CTT 肽能够抑制 Katanin 的切断。在这里，我们研究了 CTT 序列对这种抑制活性的影响。具体来说，我们研究了自然界中发现的 CTT 序列，α1A（TUBA1A）、去酪氨酸化的α1A、Δ2α1A、β5（TUBB/TUBB5）、β2a（TUBB2A）、β3（TUBB3）和β4b（TUBB4b）。我们发现这些天然的 CTT 具有不同的抑制能力，最明显的是β3 CTT 不能抑制 Katanin。两种非天然的 CTT 尾结构也不能抑制，尽管它们与α1 或β5 序列有 94%的序列同一性。令人惊讶的是，我们证明多聚 E 和多聚 D 肽能够显著抑制 Katanin。对 CTT 结构疏水性的分析表明，疏水性更强的多肽比极性更强的多肽抑制能力更低。这些实验不仅证明了抑制作用，而且还可能表明 Katanin 与各种 CTT 的相互作用和靶向性，当它们成为聚合微管丝的一部分时。
