Victor Rachel A, Altemus Jesse J, Lay Michelle A, Shepherd Sarah N, Padilla-Rodriguez Marco, Lipinski Austin, Mouneimne Ghassan, Langlais Paul R, Schwartz Jacob C
Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, 85724, USA.
Department of Pharmacology, College of Medicine, University of Arizona, Tucson, AZ, 85724, USA.
bioRxiv. 2025 Aug 31:2025.08.27.672480. doi: 10.1101/2025.08.27.672480.
Cellular biochemistry arises from various interactions between macromolecules, including proteins, nucleic acids, and lipids. These make up membrane-bound organelles, membrane-less compartments, and molecular assemblies and scaffolds. Changes due to stimuli or disease can significantly impact cell fate and metabolism. We recently reported our protocol combining crosslinking and size exclusion chromatography with mass spectrometry (SEC-MS). In this study, we explore global changes to subcellular structure in Ewing sarcoma or in response to drug treatment. Between Ewing to non-Ewing sarcoma cells, differences occur in molecular structures involved in splicing, mitochondria function, and cell division. We confirm changes to nucleoli structure. We also examine structures affected by a transcription inhibitor, flavopiridol. Following flavopiridol treatment, we observed changes to the levels of transcription and mRNA processing machinery present in large subcellular structures. Unexpected effects were also found, including structural changes to a cytoplasmic organelle, the peroxisome. Along with a reduction in peroxisome function, dissociation of peroxisome pore proteins PEX13 and PEX14 was detected by STORM microscopy. We conclude that SEC-MS combined with crosslinking is a valuable method to detect and quantify drug or disease effects on subcellular structures and may shed light on new aspects to mechanisms underlying their biologic outcomes.
细胞生物化学源于大分子之间的各种相互作用，包括蛋白质、核酸和脂质。这些大分子构成了膜结合细胞器、无膜区室以及分子组装体和支架。由于刺激或疾病引起的变化会显著影响细胞命运和新陈代谢。我们最近报道了我们将交联、尺寸排阻色谱与质谱联用（SEC-MS）的方法。在本研究中，我们探索尤因肉瘤中或药物治疗后亚细胞结构的整体变化。在尤因肉瘤细胞与非尤因肉瘤细胞之间，参与剪接、线粒体功能和细胞分裂的分子结构存在差异。我们证实了核仁结构的变化。我们还研究了受转录抑制剂黄酮哌啶醇影响的结构。黄酮哌啶醇处理后，我们观察到大型亚细胞结构中存在的转录和mRNA加工机制水平的变化。还发现了意想不到的影响，包括一种细胞质细胞器过氧化物酶体的结构变化。随着过氧化物酶体功能的降低，通过超分辨光学显微镜（STORM）检测到过氧化物酶体孔蛋白PEX13和PEX14的解离。我们得出结论，交联结合SEC-MS是一种检测和量化药物或疾病对亚细胞结构影响的有价值方法，可能会揭示其生物学结果潜在机制的新方面。
