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Protein expression in warmed human lens epithelium cells using shotgun proteomics

ABSTRACT: In previous studies, we reported that cumulative thermal dose estimation in crystalline lens, as determined through computer simulation analysis using a supercomputer and a biothermal transport equation, showed a strong correlation with the prevalence of nuclear cataracts. In this study, we have investigated what kind of protein expressed underlying normal (35.0 °C) and warming conditions (37.5 °C) by using the shotgun Liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based global proteomic approach. A total of 615 proteins were identified in the iHLEC-NY2, including 307 (49.9%) present in both lens cultured at 35.0 °C and 37.5 °C, 130 (21.1%) unique to lens cultured at 35.0 °C, and 178 (29.0%) unique to lens cultured at 37.5 °C. Furthermore, we have discussed the changes in protein expression in warmed iHLEC-NY2 cells using Gene Ontology analysis and a label-free semiquantitative method based on spectral counting. Shotgun proteomic analysis revealed that warming decreased the expression of actin, alpha cardiac muscle 1, actin-related protein 2, putative tubulin-like protein alpha-4B, ubiquitin carboxyl-terminal hydrolase 17-like protein 1, ubiquitin-ribosomal protein eL40 fusion protein, ribosome biogenesis protein BMS1 homolog, histone H2B type 1-M, and histone H2A.J. in iHLEC-NY2. Thus, the decrease in specific protein levels of actin, tubulin, ubiquitin, ribosomes, and histones may be related to cataract development under warming conditions. This study may serve as a valuable indicator for elucidating the relationship between temperature and onset of nuclear cataracts.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Noriaki Nagai

PROVIDER: PXD059029 | JPOST Repository | Fri Dec 19 00:00:00 GMT 2025

REPOSITORIES: jPOST

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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

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Protein expression in warmed human lens epithelium cells using shotgun proteomics

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