用高选择性肽HK2pep分析己糖激酶2脱离线粒体相关膜的作用

Francesco  Ciscato; Federica  Chiara; Riccardo  Filadi; Andrea   Rasola

doi:10.21769/BioProtoc.4087

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

用高选择性肽HK2pep分析己糖激酶2脱离线粒体相关膜的作用

FC Francesco Ciscato ^* email

FC Federica Chiara ^*

RF Riccardo Filadi

AR Andrea Rasola email

(*contributed equally to this work) 发布: 2021年07月20日第11卷第14期 DOI: 10.21769/BioProtoc.4087 浏览次数: 3579

评审: Julie WeidnerAnonymous reviewer(s)

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Abstract

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 Ca²⁺ overload caused by the opening of inositol-3-phosphate receptors on the endoplasmic reticulum (ER) and Ca²⁺ entry through the plasma membrane leading to Ca²⁺-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, Ca²⁺ 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.

Keywords: Hexokinase 2 (己糖激酶2)

Cell-penetrating peptide (细胞穿透肽)

Mitochondria-associated membranes (线粒体相关膜)

Ca²⁺ (Ca²⁺)

Anti-neoplastic strategy (抗肿瘤策略)

Cancer (癌症)

Mitochondria (线粒体)

Background

Metabolic rewiring in tumor cells (Boroughs and DeBerardinis, 2015; Vander Heiden and DeBerardinis, 2017) encompasses increased uptake and usage of glucose and decreased oxidative phosphorylation (OXPHOS) (Cannino et al., 2018; Faubert et al., 2020), conferring a selective advantage to neoplastic cells to survive and thrive under shortage of both nutrients and oxygen (Nakazawa et al., 2016). Hence, targeting metabolic components offers promising therapeutic perspectives, and the selective inhibition of glucose utilization has been considered for clinical cancer therapy (Hay, 2016). Hexokinases convert glucose to glucose-6-phosphate and make it available for utilization in glycolysis, the pentose phosphate pathway, glycogenesis, and hexosamine biosynthesis (Wilson, 2003). The most active isozyme, hexokinase 2 (HK2), is overexpressed in numerous types of cancer and constitutes a promising target for the development of anti-neoplastic strategies (Roberts and Miyamoto, 2015). Indeed, HK2 plays a major role in the metabolic rewiring of tumors and is induced by oncogenic K-Ras activation (Patra et al., 2013) or in response to hypoxia (Semenza, 2013). HK2 overexpression is related to stage progression, acquisition of invasive and metastatic capabilities, and poor prognosis (Mathupala et al., 2010). In tumor cells, HK2 binds to the outer mitochondrial membrane following Akt-dependent phosphorylation (Miyamoto et al., 2008), and mitochondrial binding of HK2 has been associated with the protection of cancer cells from noxious stimuli (Roberts and Miyamoto, 2015). The natural consequence of these observations was the development of therapeutic strategies aimed at inhibiting the activity of HKs; however, the clinical use of hexokinase inhibitors was hampered by the lack of specificity or the side effects (Akins et al., 2018) elicited by the conserved nature of the active sites among the ubiquitously expressed hexokinase isozymes (Roberts and Miyamoto, 2015).

We have previously exploited an alternative approach to target HK2, based on detaching it from mitochondria with a peptide corresponding to the N-terminal hydrophobic domain of the enzyme. This treatment causes the opening of a mitochondrial channel, the permeability transition pore (PTP) (Rasola and Bernardi, 2011), and consequent cell death (Chiara et al., 2008; Masgras et al., 2012; Pantic et al., 2013). Recently, we established that HK2 localizes to domains of interaction between the ER and mitochondria called mitochondria-associated membranes (MAMs), and its displacement elicits a massive Ca²⁺ flux from the ER and across the plasma membrane into mitochondria, inducing mitochondrial depolarization and death in a variety of cancer models, both in vitro and in vivo, in a calpain-dependent manner (Ciscato et al., 2020). We have improved HK2 peptide efficacy and specificity by adding a polycation stretch required for plasma membrane permeation linked to a shielding polyanion sequence through a matrix metalloprotease 2 and 9 (MMP2/9) target sequence. This novel tool, called HK2pep, is specifically taken up by neoplastic cells when its polycation sequence is unmasked by removal of the polyanion stretch through MMP2/9 cleavage in the tumor microenvironment, where these proteases are highly induced (Figure 1). HK2pep has proven to be an excellent tool to dissect the precise localization of HK2 in MAMs and the cascade of events leading to tumor cell death, in addition to being effective in tumors allografted in mice, while leaving hexokinase enzymatic activity unaffected, thus protecting healthy tissues from any off-target effects of the treatment (Ciscato et al., 2020).

Taken together, our data disclose novel signaling pathways primed by HK2 displacement from MAMs and open possibilities for the development of effective anti-neoplastic strategies, alone or in combination with other chemotherapies.

Figure 1. Modified from Ciscato et al., 2020. Functional unit composition of HK2pep. The HK2-targeting sequence is in red; the polycation and polyanion stretches are in light blue and light green, respectively; the MMP2/9 target sequence is in yellow. Cl-HK2pep is the active peptide after MMP2/9 cleavage.

Materials and Reagents

Flasks, 25 cm² (Falcon, catalog number: 353108)
6-well plates (Falcon, catalog number: 353046)
12-well plates (Falcon, catalog number: 353103)
24-well plates (Falcon, catalog number: 353047)
96-well plates (Falcon, catalog number: 353916)
13-mm diameter coverslips (VWR, catalog number: 631-0149P)
18-mm diameter coverslips (VWR, catalog number: 631-0153P)
24-mm diameter coverslips (VWR, catalog number: 631-0161)
PBS (Sigma-Aldrich, catalog number: D8537, storage RT)
Trypsin-EDTA (GIBCO, catalog number: 25200-056, storage 4°C)
NaCl (Sigma-Aldrich, catalog number: S9888, storage RT)
Trizma® base (Sigma-Aldrich, catalog number: T6066, storage RT)
EDTA (Sigma-Aldrich, catalog number: E5134, storage RT)
Triton X-100 (Sigma, catalog number: 9002-93-1, storage RT)
Glycerol (Sigma-Aldrich, catalog number: G5516, storage RT)
Pierce^TM BCA protein assay (Thermo Scientific^TM, catalog number: 23225, storage RT)
MgCl₂ (Sigma-Aldrich, catalog number: 63068, storage RT)
ATP (Sigma-Aldrich, catalog number: A2383, storage -20°C)
Glucose (Sigma-Aldrich, catalog number: G8270, storage RT)
Glucose 6-phosphate-dehydrogenase (Sigma-Aldrich, catalog number: SRP6505, storage -20°C)
NADP (Sigma-Aldrich, catalog number: 93205, storage -20°C)
SPLICS_s (split-GFP-based contact site sensor short) and SPLICS_L (split-GFP-based contact site sensor long) plasmids (Cieri et al., 2018, available from the authors) (storage -20°C)
OptiMEM (GIBCO, catalog number: 51985026, storage 4°C)
TransIT-LT1 (Mirus, catalog number: MIR 2304, storage 4°C)
KCl (Sigma-Aldrich, catalog number: P3911, storage RT)
KH₂PO₄ (Merck, catalog number: 1551139, storage RT)
CaCl₂ (Sigma-Aldrich, catalog number: 21097, storage RT)
HEPES (Sigma-Aldrich, catalog number: H3375, storage RT)
HK2pep: (Chemical synthesis, seq. MIASHLLAYFFTELN-bA-RRRRRRRRR-PLGLAG-Ahx-EEEEEEEE, storage -20°C)
SCRpep: (Chemical synthesis, seq. VGAHAGEYGAEALER-bA-RRRRRRRRR-PLGLAG-Ahx-EEEEEEEE, storage -20°C)
cl-HK2pep (Chemical synthesis, seq. MIASHLLAYFFTELN-βA-RRRRRRRRR-PLG, storage -20°C)
cl-SCRpep (Chemical synthesis, seq. VGAHAGEYGAEALER-βA-RRRRRRRRR-PLG storage -20°C). All peptides were synthesized by automatic solid-phase procedures (for details, see Ciscato et al., 2020). Suggested purity ≥ 95% measured by analytical reversed-phase HPLC. Peptides are not commercially available but can be synthesized on demand by specialized companies.
PFA (Sigma-Aldrich, catalog number: P6148, storage 4°C)
NH₄Cl (Sigma-Aldrich, catalog number: A9434, storage RT)
BSA (Sigma-Aldrich, catalog number: AG4503, storage 4°C)
Goat serum (Sigma-Aldrich, catalog number: G6767, storage -20°C)
Gelatin (Sigma-Aldrich, catalog number: G2500, storage -20°C)
Rabbit monoclonal anti-HK2 antibody (Thermo Scientific, catalog number: H.738.7, storage -20°C)
AlexaFluor555 donkey anti-rabbit IgG (Thermo Fisher Scientific, catalog number: A-31572, storage -20°C)
Mowiol 4-88 (Sigma-Aldrich, catalog number: 81381, storage RT)
Plasmids encoding mitochondria-targeted GCAMP6f (Filadi et al., 2018, available from the authors, storage -20°C)
Cyclosporin H (Vinci-Biochem, catalog number: AG-CN2-0447-M005, storage -20°C)
TetraMethyl-Rhodamine Methyl ester (TMRM) (Invitrogen, MitoProbe^TM, catalog number: M20036, storage -20°C)
Annexin V-FITC - Annexin-V-FLUOS labeling reagent (Roche - now sold by MERCK, catalog number: 11828681001, storage -20°C)
7-aminoactinomycin D (7-AAD; Sigma-Aldrich, catalog number: A9400, storage -20°C)

Equipment

Cell culture incubators (Thermo Scientific Forma^TM Steri-Cycle^TM CO₂ Incubator)
Equipped cell culture hoods (Angelantoni Industries, catalog number: VBH 48 C2)
Micropipettes (Gilson Pipetman Classic, P1,000, P200, P20, P10, P2)
Burker chamber (VWR, catalog number: HECH40443703)
Centrifuges from 200 to 2,000 × g, fixed bucket, no specific rotors needed (e.g., MPW Med. Instruments, catalog number: MPW 251)
Thermo-shaker (Biosan, catalog number: TS-100)
Centrifuges from 200 to 16,000 × g, fixed bucket, no specific rotors needed (e.g., Eppendorf, catalog number: 5417 R)
Plate-Reader Spectrophotometer (TECAN, model: Infinite M200 spectrophotometer)
Confocal microscope (Leica SP5-II equipped with a 100×/1.4 N.A. plan apochromat objective, a WLL laser to excite each specific dye, and a HyD detector for signal collection)
Inverted fluorescence microscope (Zeiss Axiovert 100, Fluar 40× oil objective, NA 1.30)
Inverted fluorescence microscope (Leica, model: DMI6000 B)
Cytofluorimeter (BD FACSCanto^TM II)

Software

i-control^TM(for TECAN, Infinite M200 spectrophotometer, https://lifesciences.tecan.com/plate_readers/infinite_200_pro?p=tab--3)
Excel data analysis software (Microsoft Office)
Leica Application Suite LAS-AF (for Leica SP5-II https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp5-ii)
ImageJ (NIH, https://imagej.nih.gov/ij/download.html)
For the Zeiss microscope, synchronization of the cool camera with the excitation source is performed by a custom-made software package (Roboscope - developed by Catalin Dacian Ciubotaru at VIMM, Padova, Italy); alternatively, the specific manufacturer’s microscope software is suitable for kinetic image acquisition
Leica Application Suite LAS-AF for the Leica fluorescence microscope (https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/)
BD FACSDiva^TM for the BD FACSCanto^TM II cytoflurimeter (https://www.bdbiosciences.com/en-us/instruments/research-instruments/research-software/flow-cytometry-acquisition/facsdiva-software)

Procedure

English

中文翻译

文章信息

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如何引用

Ciscato, F., Chiara, F., Filadi, R. and Rasola, A. (2021). Analysis of the Effects of Hexokinase 2 Detachment From Mitochondria-Associated Membranes with the Highly Selective Peptide HK2pep. Bio-protocol 11(14): e4087. DOI: 10.21769/BioProtoc.4087.