Bourne Christopher M, Raniszewski Nicole R, Kulkarni Madhura, Exconde Patrick M, Liu Sherry, Yost Winslow, Wrong Tristan J, Patio Robert C, Mahale Ashutosh, Kardhashi Matilda, Shosanya Teni, Kambayashi Mirai, Discher Bohdana M, Brodsky Igor E, Burslem George M, Taabazuing Cornelius Y
Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
Department of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104, USA.
bioRxiv. 2025 Feb 27:2025.02.23.639785. doi: 10.1101/2025.02.23.639785.
Caspases are a family of cysteine proteases that act as molecular scissors to cleave substrates and regulate biological processes such as programmed cell death and inflammation. Extensive efforts have been made to identify caspase substrates and to determine factors that dictate substrate specificity. We recently discovered that that the human inflammatory caspases (caspases-1, -4, and -5) cleave the cytokines IL-1β and IL-18 in a sequence-dependent manner. Here, we report the development of a new peptide-based probe and inhibitor based on the tetrapeptide sequence of IL-18 (LESD). We found that this inhibitor was most selective and potent at inhibiting caspase-8 activity (IC = 50 nM). We also discovered that our LESD-based inhibitor is more potent than the currently used z-IETD-FMK inhibitor that is thought to be the most selective and potent inhibitor of caspase-8. Accordingly, we demonstrate that the LESD based inhibitor prevents caspase-8 activation during infection in primary bone-marrow derived macrophages. Furthermore, we characterize the selectivity and potency of currently known substrates and inhibitors for the apoptotic and inflammatory caspases using the same activity units of each caspase. Our findings reveal that VX-765, a known caspase-1 inhibitor, also inhibits caspase-8 (IC = 1 μM) and even when specificities are shared, the caspases have different efficiencies and potencies for shared substrates and inhibitors. Altogether, we report the development of new tools that will facilitate the study of caspases and their roles in biology.
半胱天冬酶是一类半胱氨酸蛋白酶，它们就像分子剪刀一样切割底物，并调节诸如程序性细胞死亡和炎症等生物学过程。人们已经付出了巨大努力来鉴定半胱天冬酶底物，并确定决定底物特异性的因素。我们最近发现，人类炎性半胱天冬酶（半胱天冬酶-1、-4和-5）以序列依赖性方式切割细胞因子IL-1β和IL-18。在此，我们报告了一种基于IL-18（LESD）四肽序列的新型肽基探针和抑制剂的研发情况。我们发现这种抑制剂在抑制半胱天冬酶-8活性方面具有最高的选择性和效力（IC = 50 nM）。我们还发现，我们基于LESD的抑制剂比目前使用的z-IETD-FMK抑制剂更有效，后者被认为是半胱天冬酶-8最具选择性和效力的抑制剂。因此，我们证明基于LESD的抑制剂可在原代骨髓来源的巨噬细胞感染期间阻止半胱天冬酶-8的激活。此外，我们使用每种半胱天冬酶的相同活性单位来表征目前已知的凋亡和炎性半胱天冬酶底物及抑制剂的选择性和效力。我们的研究结果表明，已知的半胱天冬酶-1抑制剂VX-765也能抑制半胱天冬酶-8（IC = 1 μM），而且即使存在共享的特异性，半胱天冬酶对共享底物和抑制剂的效率和效力也有所不同。总之，我们报告了新工具的研发情况，这将有助于对半胱天冬酶及其在生物学中的作用进行研究。
