Lacham-Hartman Shiran, Oren Ofek, Rabinovitch Maya, Papo Niv
Avram and Stella Goldstein-Goren Department of Biotechnology Engineering and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.
Int J Biol Macromol. 2025 Aug;320(Pt 4):146066. doi: 10.1016/j.ijbiomac.2025.146066. Epub 2025 Jul 16.
Amyloid beta 42 (Aβ42) and amyloid precursor protein inhibitor (APPI) are amyloid precursor protein (APP) domains. As fragments, Aβ42 is neurotoxic and APPI inhibits kallikrein-related peptidase 6 (KLK6). While KLK6-dependent cleavage of APP695 (an APP isoform that lacks APPI) has been shown to result in Aβ production, its proteolytic activity on APPI-containing isoforms (APP751 and APP770) and the molecular function of APPI in APP proteolysis have not been explored. In addressing this knowledge gap, we show that APP695 serves as a KLK6 substrate in SH-SY5Y neuroblastoma cells. We also show that APPI, as either a soluble fragment or an APP751 domain, strongly inhibits KLK6 catalytic activity and reduces KLK6-mediated APP695 cleavage in a dose-dependent manner. Moreover, the soluble extracellular APPI fragment reduces Aβ42 aggregation and fibril length, thereby favoring the formation of Aβ42 oligomers that cannot permeate into cells and thus significantly reducing apoptosis in SH-SY5Y neuroblastoma cells. In a similar way, the expression of both intracellular APPI and Aβ42 (vs. Aβ42 alone) reduces Aβ42 aggregate formation and increases neuronal cell viability. Our results provide an explanation for the presence of alternatively spliced APP isoforms in the brains of patients with Alzheimer's disease (AD) and reveal the roles of APPI in APP cleavage and in the formation, aggregation and toxicity of both extra- and intracellular Aβ peptide fragments. Our results suggest that APPI may serve as a dual inhibitor, disrupting both KLK6 proteolytic activity and Aβ42 aggregation, thereby offering a potential therapeutic agent for reducing amyloid beta toxicity in AD.
淀粉样β蛋白42（Aβ42）和淀粉样前体蛋白抑制剂（APPI）是淀粉样前体蛋白（APP）的结构域。作为片段，Aβ42具有神经毒性，而APPI可抑制激肽释放酶相关肽酶6（KLK6）。虽然已表明KLK6依赖性切割APP695（一种缺乏APPI的APP异构体）会导致Aβ生成，但其对含APPI异构体（APP751和APP770）的蛋白水解活性以及APPI在APP蛋白水解中的分子功能尚未得到研究。为填补这一知识空白，我们发现APP695在SH-SY5Y神经母细胞瘤细胞中是KLK6的底物。我们还表明，APPI作为可溶性片段或APP751结构域，能强烈抑制KLK6的催化活性，并以剂量依赖性方式减少KLK6介导的APP695切割。此外，可溶性细胞外APPI片段可减少Aβ42的聚集和纤维长度，从而有利于形成无法渗透到细胞内的Aβ42寡聚体，进而显著减少SH-SY5Y神经母细胞瘤细胞中的细胞凋亡。同样，细胞内APPI和Aβ42（与单独的Aβ42相比）的表达均可减少Aβ42聚集体的形成并提高神经元细胞活力。我们的研究结果解释了阿尔茨海默病（AD）患者大脑中存在可变剪接的APP异构体的原因，并揭示了APPI在APP切割以及细胞外和细胞内Aβ肽片段的形成、聚集和毒性中的作用。我们的研究结果表明，APPI可能作为一种双重抑制剂，破坏KLK6的蛋白水解活性和Aβ42的聚集，从而为降低AD中淀粉样β蛋白毒性提供一种潜在的治疗药物。
