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. 2016 Jul 1;291(27):13905-13916.
doi: 10.1074/jbc.M116.731281. Epub 2016 May 3.

Cathepsin Protease Controls Copper and Cisplatin Accumulation via Cleavage of the Ctr1 Metal-binding Ectodomain

Helena Öhrvik  1 , Brandon Logeman  2 , Boris Turk  3 , Thomas Reinheckel  4 , Dennis J Thiele  5
Affiliations

Cathepsin Protease Controls Copper and Cisplatin Accumulation via Cleavage of the Ctr1 Metal-binding Ectodomain

Helena Öhrvik et al. J Biol Chem. .

Abstract

Copper is an essential metal ion for embryonic development, iron acquisition, cardiac function, neuropeptide biogenesis, and other critical physiological processes. Ctr1 is a high affinity Cu(+) transporter on the plasma membrane and endosomes that exists as a full-length protein and a truncated form of Ctr1 lacking the methionine- and histidine-rich metal-binding ectodomain, and it exhibits reduced Cu(+) transport activity. Here, we identify the cathepsin L/B endolysosomal proteases functioning in a direct and rate-limiting step in the Ctr1 ectodomain cleavage. Cells and mice lacking cathepsin L accumulate full-length Ctr1 and hyper-accumulate copper VSports手机版. As Ctr1 also transports the chemotherapeutic drug cisplatin via direct binding to the ectodomain, we demonstrate that the combination of cisplatin with a cathepsin L/B inhibitor enhances cisplatin uptake and cell killing. These studies identify a new processing event and the key protease that cleaves the Ctr1 metal-binding ectodomain, which functions to regulate cellular Cu(+) and cisplatin acquisition. .

Keywords: anti-cancer drug; cathepsin; cisplatin; copper transport; cysteine protease; intracellular processing; metal homeostasis; protein processing V体育安卓版. .

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Figures

FIGURE 1.
FIGURE 1.
Ctr1 ectodomain cleavage occurs in a Ctr2-stimulated, cysteine protease-dependent manner. A, model showing monomeric full-length Ctr1 and tCtr1, with Met and His residues in the ectodomain indicated in blue and red, respectively, and glycans indicated with branches. B, cells containing a doxycycline-inducible Ctr2 (Tet-On Ctr2) were treated with 100 ng/ml doxycycline for the times indicated, harvested, and immunoblotted with anti-Ctr2, anti-Ctr1 (T, truncated; F, full-length), and anti-tubulin antibody. C, MEFs were treated with DMSO, 2 μg/ml TIMP-2, TIMP-3, and TAPI-2 or 5 μm BB-94 for 16 h, and protein extract was analyzed by immunoblotting with anti-Ctr1 (T, truncated; F, full-length) and anti-tubulin antibody. D, MEFs were treated with 10 μm E64d, and CHO and HEK cells were treated with 50 μm E64d for 16 h before immunoblot analysis as in B. E, Tet-On Ctr2 cells were cultured with or without 100 ng/ml doxycycline for 24 h before treatment with either DMSO or E64d (50 μm), and protein extracts were analyzed with immunoblotting as in B.
FIGURE 2.
FIGURE 2.
Cathepsin L inhibition results in decreased Ctr1 ectodomain cleavage and increased copper accumulation. A, MEFs were harvested and processed for endolysosomal isolation (see “Experimental Procedures”). Samples were resolved via SDS-PAGE and analyzed by immunoblotting with anti-Ctr2, anti-Ctr1 (T, truncated; F, full-length), anti-cathepsin L, anti-cathepsin B, anti-Lamp1, and anti-Cox IV. N, nuclear fraction; S, soluble fraction; 1, 2, and 3 represent fractions collected from the iodixanol gradient after ultracentrifugation. B, total protein extracts were isolated from wild type (WT); cathepsin B−/− and cathepsin (Cath) L−/− MEFs were analyzed by immunoblotting as in A. C, cathepsin L−/−B−/− cells were transfected with a plasmid expressing cathepsin L and cultured for 48 h. Total protein extracts were resolved by SDS-PAGE and analyzed by immunoblotting alongside WT, cathepsin L−/−, and cathepsin L−/−B−/− cells. D, MEFs were treated with DMSO, 10 μm cathepsin B (CA074Me), or 10 μm cathepsin L inhibitors (Inhib) for 16 h and analyzed by immunoblotting as in B. Shown are lanes cut from the same membrane with the same x-ray film exposure time. E and F, cells were treated with 30 μm indicated inhibitor and 100 μm bathocuproinedisulfonic acid for 16 h before switching to media containing 5 μm copper and fresh inhibitor before harvesting and ICP-MS analysis of total cellular copper. Data are presented as mean ± S.D. from four biological replicates. *, p ≤ 0.05.
FIGURE 3.
FIGURE 3.
Contribution of cathepsin B and Ctr2 to the processing of Ctr1. A, wild type (cathepsin L+/+) and cathepsin L−/− MEFs were treated with DMSO or 10 μm E64d inhibitor for 16 h. Total protein extract was resolved by SDS-PAGE and immunoblotted with anti-Ctr1 and anti-tubulin antibody. B, wild type (cathepsin L+/+) and cathepsin L−/− MEFs were treated with 25 nm scrambled (Sc) RNA or siRNA directed against Ctr2 for 72 h. Total protein extracts were immunoblotted with anti-Ctr1, anti-Ctr2, and anti-tubulin antibody. C, total protein extracts from wild type (cathepsin L+/+) and cathepsin L−/− MEFs, resolved by SDS-PAGE, and analyzed by immunoblotting with anti-cathepsin B antibody. Cath, cathepsin. GAPDH as loading control. D, immunoblotting of wild type (Ctr2+/+) and Ctr2−/− MEF protein extracts derived from endolysosomal enrichment (see “Experimental Procedures”). Blots were analyzed by probing with anti-Ctr2, anti-Ctr1 (T, truncated; F, full-length), anti-cathepsin L, anti-cathepsin B, anti-Lamp1, and anti-CoxIV. N, nuclear fraction; S, soluble fraction; 1, 2, and 3 represent fractions collected from the iodixanol gradient after ultracentrifugation.
FIGURE 4.
FIGURE 4.
Cathepsin L cleaves the CTR1 ectodomain in vitro and primes further ectodomain processing. A, diagram of the CTR1 ectodomain purified for in vitro cathepsin cleavage studies and SDS-PAGE of purified ectodomain (Ctr1 ecto). Lane M, molecular mass marker. B, CTR1 ectodomain was incubated with recombinant cathepsin L at the indicated enzyme concentration, and products were resolved by SDS-PAGE and Ponceau S staining. The products generated by cathepsin L cleavage are indicated as 1 or 2. C, site of cathepsin L cleavage (red arrowhead) determined by mass spectrometry and the previously identified amino termini of tCtr1 (blue arrowhead). Asterisks indicate residues within the cathepsin L cleavage site found in several mammalian Ctr1 proteins. D, Ctr1−/−Ctr2−/− cells containing a Dox-inducible Ctr2 gene were transfected with either an empty vector (V), a vector containing wild type Ctr1 (WT), or Ctr1 lacking the first 8 residues (Ctr1Δ1–8) before being treated with 100 ng/ml doxycycline for 48 h. Cells were then harvested and processed for immunoblotting with the indicated antibodies.
FIGURE 5.
FIGURE 5.
Cathepsin L−/− mice exhibit defects in Ctr1 ectodomain cleavage and accumulate copper. A, 2-month-old wild type (cathepsin L+/+) and cathepsin L−/− mice were analyzed for copper levels by ICP-MS in brain (Br), liver (Li), kidney (Ki), spleen (Sp), lymph nodes (Ly), and testis (Te). Note different scales for copper levels in different tissues. B, tissue zinc analysis as in A. Data are presented as means ± S.D. from three to four mice, *, p ≤ 0.05; **, p ≤ 0.01. C, protein extracts from the indicated tissues from WT (+/+) and cathepsin L−/− (Cath L−/−) littermates were immunoblotted with anti-Ctr1 and anti-SOD1 antibody, with cathepsin L genotypes indicated. Shown are the full-length (F) and truncated (T) forms of Ctr1. A short exposure shows the abundance of tCtr1 and a longer exposure reveals the expression of both full-length and truncated Ctr1.
FIGURE 6.
FIGURE 6.
Cathepsin L inhibition of Ctr1 ectodomain cleavage enhances cisplatin accumulation and efficacy in vitro. A, Ctr1−/− MEFs were transfected with vector control (v) or vector containing human Ctr1 (hCtr1) and treated either with DMSO or 10 μm cathepsin L inhibitor for 16 h followed by a pulse of 200 μm cisplatin for 2 h. Cells were harvested and total cellular platinum (Pt) levels measured by ICP-MS and normalized to protein concentrations. B, wild type or Ctr1−/− MEFs were pre-treated with DMSO or 10 μm E64d for 2 h followed by DMSO or 50 μm cisplatin for 12 h, and cell viability was assessed by recording the formation of the fluorescent product resorufin, with normalization against vehicle-treated cells. Data are presented as mean ± S.D. from three to four biological replicates. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. C, model for the Ctr2 and cathepsin L (Cath L)-mediated cleavage of the Ctr1 ectodomain to generate tCtr1, within an endolysosomal compartment. In this model, we envision two mechanisms by which Ctr2 and cathepsin L/B cooperate in Ctr1 ectodomain cleavage; [1] binding of Ctr2 to Ctr1 may provoke a conformational switch in the Ctr1 ectodomain, providing access by cathepsin L/B, or [2] Ctr2 may recruit cathepsin L/B and deliver the protease to the ectodomain cleavage site.
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