Chatterjee Tanaya, Mukherjee Rimi, Das Gaurav, Pal Arumay, Bag Sudipta, Chatterjee Barun K, Chakrabarti Pinak
Department of Biological Science, Bose Institute, EN Block, Sector V, Kolkata 700091, India.
Department of Signal Transduction & Biogenic Amines, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road, Kolkata 700026, India.
Biochemistry. 2025 Jul 1;64(13):2765-2777. doi: 10.1021/acs.biochem.5c00087. Epub 2025 Jun 20.
Microtubules, the key component of the cytoskeleton, are indispensable for various cellular tasks such as cell differentiation, mitosis, etc. Disruption of microtubule assembly plays a crucial role in tumor regulation. Protein l-isoaspartyl methyltransferase (PIMT), found in abundance in brain, is known to repair abnormal isoaspartate residues, formed on aging, to the normal aspartate. Using various biophysical techniques, we show that PIMT can compromise the microtubule network after internalization in cells. The binding of PIMT to tubulin overlaps with the vinblastine binding site, as inferred from the competitive assay. Experiments using MCF-7 breast cancer cells revealed the binding of PIMT with intracellular tubulin and the disruption of the network. Results are reproducible in an additional breast cancer cell line, MDA-MB-231. In vivo experiments using mice with breast cancer cells revealed tumor regression after treatment with PIMT. Like other antimitotic agents, PIMT can target tubulin, regulating the structure and dynamics of microtubules. The free energy of binding of PIMT to tubulin was found to be -6.3 kcal/mol obtained from Isothermal Titration Calorimetry (ITC). The PIMT-tubulin complex structure determined by AlphaFold suggests an interface that overlays considerably with that between two tubulin heterodimers in the microtubule, suggesting a mechanism by which the binding of PIMT can induce the dissociation of the microtubule. Asp/Asn residues present at the latter interface would be subjected to various degrees of isomerization and thereby control the properties and functions of the microtubule and also be a substrate for the repair enzyme, PIMT.
微管作为细胞骨架的关键组成部分，对于诸如细胞分化、有丝分裂等各种细胞任务而言不可或缺。微管组装的破坏在肿瘤调控中起着至关重要的作用。在大脑中大量存在的蛋白质L-异天冬氨酰甲基转移酶（PIMT），已知可将衰老过程中形成的异常异天冬氨酸残基修复为正常的天冬氨酸。运用各种生物物理技术，我们发现PIMT在细胞内化后会破坏微管网络。从竞争试验推断，PIMT与微管蛋白的结合与长春碱结合位点重叠。使用MCF-7乳腺癌细胞进行的实验揭示了PIMT与细胞内微管蛋白的结合以及网络的破坏。在另一种乳腺癌细胞系MDA-MB-231中，结果具有可重复性。使用携带乳腺癌细胞的小鼠进行的体内实验表明，用PIMT治疗后肿瘤会消退。与其他抗有丝分裂剂一样，PIMT可靶向微管蛋白，调节微管的结构和动力学。通过等温滴定量热法（ITC）测得PIMT与微管蛋白结合的自由能为-6.3千卡/摩尔。由AlphaFold确定的PIMT-微管蛋白复合物结构表明，其界面与微管中两个微管蛋白异二聚体之间的界面有相当大的重叠，这表明PIMT的结合可诱导微管解离的一种机制。存在于后一界面的天冬氨酸/天冬酰胺残基会发生不同程度的异构化，从而控制微管的特性和功能，并且还是修复酶PIMT的底物。
