Analysis of the Effects of Hexokinase 2 Detachment From Mitochondria-Associated Membranes with the Highly Selective Peptide HK2pep.

Autor:	Ciscato F; Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy., Chiara F; Department of Surgery, Oncology and Gastroenterology (DISCOG), University of Padova, Padova, Italy., Filadi R; Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy.; Neuroscience Institute, Italian National Research Council (CNR), Padova, Italy., Rasola A; Department of Biomedical Sciences (DSB), University of Padova, Padova, Italy.
Jazyk:	angličtina
Zdroj:	Bio-protocol [Bio Protoc] 2021 Jul 20; Vol. 11 (14), pp. e4087. Date of Electronic Publication: 2021 Jul 20 (Print Publication: 2021).
DOI:	10.21769/BioProtoc.4087
Abstrakt:	The crucial role of hexokinase 2 (HK2) in the metabolic rewiring of tumors is now well established, which makes it a suitable target for the design of novel therapies. However, hexokinase activity is central to glucose utilization in all tissues; thus, enzymatic inhibition of HK2 can induce severe adverse effects. In an effort to find a selective anti-neoplastic strategy, we exploited an alternative approach based on HK2 detachment from its location on the outer mitochondrial membrane. We designed a HK2-targeting peptide named HK2pep, corresponding to the N-terminal hydrophobic domain of HK2 and armed with a metalloprotease cleavage sequence and a polycation stretch shielded by a polyanion sequence. In the tumor microenvironment, metalloproteases unleash polycations to allow selective plasma membrane permeation in neoplastic cells. HK2pep delivery induces the detachment of HK2 from mitochondria-associated membranes (MAMs) and mitochondrial Ca 2+ overload caused by the opening of inositol-3-phosphate receptors on the endoplasmic reticulum (ER) and Ca 2+ entry through the plasma membrane leading to Ca 2+ -mediated calpain activation and mitochondrial depolarization. As a result, HK2pep rapidly elicits death of diverse tumor cell types and dramatically reduces in vivo tumor mass. HK2pep does not affect hexokinase enzymatic activity, avoiding any noxious effect on non-transformed cells. Here, we make available a detailed protocol for the use of HK2pep and to investigate its biological effects, providing a comprehensive panel of assays to quantitate both HK2 enzymatic activity and changes in mitochondrial functions, Ca 2+ flux, and cell viability elicited by HK2pep treatment of tumor cells. Graphical abstract: Flowchart for the analysis of the effects of HK2 detachment from MAMs. Competing Interests: Competing interests A patent application (Italian patent No. IT102019000002321, priority: 18/02/2019; PCT: PCT/IB2020/051329) has been filed by the University of Padova for the use of the HK2-targeting peptide described in this manuscript as an anti-neoplastic tool in in vitro and in vivo experiments. (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)
Databáze:	MEDLINE
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