Ac-Ile-Glu-Pro-Asp-pNA (Ac-IEPD-pNA) is a colorimetric substrate for granzyme B (GzmB) and caspase-8 [1, 2]. Granzyme B and caspase-8 preferentially bind and cleave the Ile-Glu-Pro-Asp (IEPD) peptide sequence, and the catalytic activity of the enzyme is indirectly measured by detecting the release of p-nitroaniline (pNA), which is measured by the optical density (OD) at 405 nm[3]. Granzyme B is a serine protease found in cytotoxic T cells and NK cells, which cleaves and activates several caspases involved in apoptosis[4]. Caspase-8 is a caspase protein that participates in the signal transduction of death receptors of the tumor necrosis factor receptor family and is also essential for the induction of the transcription factor NF-κB[5]. Ac-Ile-Glu-Pro-Asp-pNA is recommended as a potential granzyme B target and the optimal P4-P1 substrate sequence for granzyme B.
The structural and group characteristics of Ac-Ile-Glu-Pro-Asp-pNA are as follows:
(1) N-acetyl group (Ac): protects the N-terminus of the amino acid to prevent nonspecific reactions.
(2) Amino acid sequence (Ile-Glu-Pro-Asp): This specific sequence mimics certain natural substrates and helps to study the specificity of proteases for different substrates.
(3) p-Nitroaniline (pNA): It is a common chromogenic group that releases a yellow product with light-absorbing properties after hydrolysis, which is convenient for monitoring enzyme reactions through spectral measurement.
References:
[1]Ewen C, Kane K P, Shostak I, et al. A novel cytotoxicity assay to evaluate antigen-specific CTL responses using a colorimetric substrate for Granzyme B[J]. Journal of immunological methods, 2003, 276(1-2): 89-101.
[2]Almeida S, Domingues A, Rodrigues L, et al. FK506 prevents mitochondrial-dependent apoptotic cell death induced by 3-nitropropionic acid in rat primary cortical cultures[J]. Neurobiology of disease, 2004, 17(3): 435-444.
[3]Chaves-Pozo E, Valero Y, Lozano M T, et al. Fish granzyme A shows a greater role than granzyme B in fish innate cell-mediated cytotoxicity, Front. Immunol. 10 (2019) 2579[J]. 2019.
[4]Rousalova I, Krepela E. Granzyme B-induced apoptosis in cancer cells and its regulation[J]. International journal of oncology, 2010, 37(6): 1361-1378.
[5]Chun H J, Zheng L, Ahmad M, et al. Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency[J]. Nature, 2002, 419(6905): 395-399.
Ac-Ile-Glu-Pro-Asp-pNA（Ac-IEPD-pNA）是颗粒酶B（Granzyme B; GzmB）和caspase-8的比色底物[1, 2]。颗粒酶B和caspase-8优先结合并切割 Ile-Glu-Pro-Asp（IEPD）肽序列，通过检测对硝基苯胺（pNA）的释放来间接测量酶的催化活性，pNA的释放在405nm的光密度（OD）来测量[3]。颗粒酶B是一种在细胞毒性T细胞和NK细胞中发现的丝氨酸蛋白酶，它裂解并激活几种参与细胞凋亡的半胱天冬酶[4]。Caspase-8是一种半胱天冬酶蛋白，可参与肿瘤坏死因子受体家族死亡受体的信号转导，对于诱导转录因子NF-κB也至关重要[5]。建议将Ac-Ile-Glu-Pro-Asp-pNA作为潜在的颗粒酶B靶点和颗粒酶B的最佳P4-P1底物序列。
Ac-Ile-Glu-Pro-Asp-pNA的结构和基团特点如下：
（1）N-乙酰基（Ac）：保护氨基酸的N-末端，防止非特异性反应。
（2）氨基酸序列（Ile-Glu-Pro-Asp）：这一特定序列模仿了某些天然底物，有助于研究蛋白酶对不同底物的特异性。
（3）对硝基苯胺（pNA）：是一个常见的色原基团，水解后释放出具有吸光特性的黄色产物，方便通过光谱测定监测酶反应。