Dataset Information

GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

ABSTRACT: 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.

SUBMITTER: Quigley EB

PROVIDER: S-EPMC10970285 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Publications

GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

Quigley Elizabeth B EB DeVore Stanley B SB Khan Shaihla A SA Geisterfer Zachary M ZM Rothfuss Heather M HM Sequoia Ari O AO Thompson Paul R PR Gatlin Jesse C JC Cherrington Brian D BD Navratil Amy M AM

International journal of molecular sciences 20240310 6

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 ide ...[more]

PMID: 38542155

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Project description:Abstract: Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. It is well established that a gonadotropin releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope derived LBT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterize the temporal dynamics of this modification. Our results show that actin and tubulin are rapidly citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. Data shows that 10 nM GnRHa induces citrullination of beta-actin with maximal levels occurring at 10 minutes. The level of beta-actin citrullination is reduced in the presence of the pan-PAD inhibitor bi-phenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa induced actin reorganization in dispersed mouse gonadotrope cells. GnRHa induces citrullination of beta-tubulin with elevated levels occurring at 30 minutes; similarly to beta-actin, this response is attenuated in the presence of BB-ClA. To examine the functional consequence of beta-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 LBT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that MT average lifetime increases following 30 minutes 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. Keywords: gonadotrope, peptidylarginine deiminase, citrullination, cytoskeleton, beta tubulin, beta actin, microtubules

Dataset Information

GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

Publications

GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

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