Attia Sara Aly, Truong Anh Tan, Phan Alvin, Lee Shin-Jae, Abanmai Manal, Markanovic Marinella, Avila Hugo, Luo Haozhong, Ali Atham, Sreekumar Parameswaran G, Kannan Ram, MacKay J Andrew
Department of Pharmacology and Pharmaceutical Sciences, Mann School of Pharmacy and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA.
Mann Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, CA 90089, USA.
Antioxidants (Basel). 2023 Sep 30;12(10):1817. doi: 10.3390/antiox12101817.
Oxidative stress-induced retinal degeneration is among the main contributing factors of serious ocular pathologies that can lead to irreversible blindness. αB-crystallin (cry) is an abundant component of the visual pathway in the vitreous humor, which modulates protein and cellular homeostasis. Within this protein exists a 20 amino acid fragment (mini-cry) with both chaperone and antiapoptotic activity. This study fuses this mini-cry peptide to two temperature-sensitive elastin-like polypeptides (ELP) with the goal of prolonging its activity in the retina.
The biophysical properties and chaperone activity of cry-ELPs were confirmed by mass spectrometry, cloud-point determination, and dynamic light scattering 'DLS'. For the first time, this work compares a simpler ELP architecture, cry-V96, with a previously reported ELP diblock copolymer, cry-SI. Their relative mechanisms of cellular uptake and antiapoptotic potential were tested using retinal pigment epithelial cells (ARPE-19). Oxidative stress was induced with HO and comparative internalization of both cry-ELPs was made using 2D and 3D culture models. We also explored the role of lysosomal membrane permeabilization by confocal microscopy.
The results indicated successful ELP fusion, cellular association with both 2D and 3D cultures, which were enhanced by oxidative stress. Both constructs suppressed apoptotic signaling (cleaved caspase-3); however, cry-V96 exhibited greater lysosomal escape.
ELP architecture is a critical factor to optimize delivery of therapeutic peptides, such as the anti-apoptotic mini-cry peptide; furthermore, the protection of mini-cry via ELPs is enhanced by lysosomal membrane permeabilization.
氧化应激诱导的视网膜变性是导致不可逆失明的严重眼部疾病的主要促成因素之一。αB-晶状体蛋白（cry）是玻璃体液中视觉通路的丰富成分，可调节蛋白质和细胞内稳态。在这种蛋白质中存在一个具有伴侣和抗凋亡活性的20个氨基酸片段（微型cry）。本研究将这种微型cry肽与两种温度敏感的弹性蛋白样多肽（ELP）融合，目的是延长其在视网膜中的活性。
通过质谱、浊点测定和动态光散射（DLS）确认cry-ELP的生物物理性质和伴侣活性。这项工作首次将更简单的ELP结构cry-V96与先前报道的ELP二嵌段共聚物cry-SI进行了比较。使用视网膜色素上皮细胞（ARPE-19）测试了它们相对的细胞摄取机制和抗凋亡潜力。用HO诱导氧化应激，并使用2D和3D培养模型对两种cry-ELP的相对内化进行比较。我们还通过共聚焦显微镜探索了溶酶体膜通透性的作用。
结果表明ELP融合成功，细胞与2D和3D培养物均有结合，氧化应激增强了这种结合。两种构建体均抑制凋亡信号（裂解的caspase-3）；然而，cry-V96表现出更大的溶酶体逃逸。
ELP结构是优化治疗性肽（如抗凋亡微型cry肽）递送的关键因素；此外，通过溶酶体膜通透性增强了ELP对微型cry的保护作用。
