Department of Zoology and Physiology, University of Wyoming, Laramie, WY 82071, USA.
Department of Pediatrics, University of Cincinnati College of Medicine, Division of Asthma Research, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
Int J Mol Sci. 2024 Mar 10;25(6):3181. doi: 10.3390/ijms25063181.
Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LβT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of β-actin, with elevated levels occurring at 10 min. The level of β-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of β-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of β-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LβT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.
肽基精氨酸脱亚氨酶（PADs 或 PADIs）催化带正电荷的精氨酸转化为中性瓜氨酸，从而改变靶蛋白的结构和功能。我们之前的工作已经证实，促性腺激素释放激素激动剂（GnRHa）刺激 PAD2 催化组蛋白瓜氨酸化，从而在促性腺激素细胞系 LβT2 中转录后调控促性腺激素基因的表达。然而，PAD 也存在于细胞质中。鉴于此，我们使用质谱（MS）鉴定了 GnRHa 刺激后发生瓜氨酸化的其他非组蛋白，并对这种修饰的时间动态进行了表征。我们的结果表明，肌动蛋白和微管蛋白发生了瓜氨酸化，这使我们假设 GnRHa 可能诱导它们的瓜氨酸化来调节细胞骨架的动力学和结构。数据表明，10 nM GnRHa 诱导β-肌动蛋白发生瓜氨酸化，10 分钟时出现高水平。泛 PAD 抑制剂联苯-苯并咪唑-Cl-酰胺（BB-ClA）的存在降低了β-肌动蛋白的瓜氨酸化水平，同时也阻止了 GnRHa 诱导的分散鼠促性腺细胞中的肌动蛋白重排。GnRHa 诱导β-微管蛋白发生瓜氨酸化，30 分钟时出现高水平，而 PAD 抑制会减弱这种反应。为了研究β-微管蛋白瓜氨酸化的功能后果，我们利用荧光标记的末端结合蛋白 1（EB1-GFP）实时追踪转染的 LβT2 细胞中微管（MT）的生长正端。EB1-GFP 的延时共聚焦显微镜显示，GnRHa 处理 30 分钟后 MT 的平均寿命增加，但 PAD 抑制会减弱这种增加。总之，我们的数据表明，GnRHa 诱导的瓜氨酸化改变了促性腺细胞中的肌动蛋白重排和 MT 寿命。
