Endocytosis of transferrin Alexa Fluor 555 conjugate is abrogated by cathepsin X.

<p>Clathrin-mediated endocytosis of transferrin Alexa Fluor 555 conjugate was followed by flow cytometry. Cathepsin X in cells was silenced with its specific siRNA, control cells were transfected with control siRNA. Mean values of three independent experiments (each in duplicate) are shown. In a control experiment transferrin receptor was saturated with holo-transferrin. *P<0.05.</p

Cathepsin X Cleaves Profilin 1 C-Terminal Tyr139 and Influences Clathrin-Mediated Endocytosis

<div><p>Cathepsin X, a cysteine carboxypeptidase, is upregulated in several types of cancer. Its molecular target in tumor cells is profilin 1, a known tumor suppressor and regulator of actin cytoskeleton dynamics. Cathepsin X cleaves off the C-terminal Tyr139 of profilin 1, affecting binding of poly-L-proline ligands and, consequently, tumor cell migration and invasion. Profilin 1 with mutations at the C-terminus, transiently expressed in prostate cancer cells PC-3, showed that Tyr139 is important for proper function of profilin 1 as a tumor suppressor. Cleaving off Tyr139 prevents the binding of clathrin, a poly-L-proline ligand involved in endocytosis. More profilin 1—clathrin complexes were present in PC-3 cells when cathepsin X was inhibited by its specific inhibitor AMS36 or silenced by siRNA. As a consequence, the endocytosis of FITC-labeled dextran and transferrin conjugate was significantly increased. These results constitute the first report of the regulation of clathrin-mediated endocytosis in tumor cells through proteolytic processing of profilin 1.</p></div

Endocytosis of fluorescein-labeled dextran is abrogated by cathepsin X.

<p>Clathrin-mediated endocytosis of fluorescein-labeled dextran (10 kDa) was followed by flow cytometry. Cathepsin X in cells was (A) treated with cathepsin X inhibitor AMS36 (10 μM) or (B) silenced with its specific siRNA. Control cells were treated with DMSO or transfected with control siRNA. (C) Prior to the inhibitor treatment, cells were treated with 10 μM chlorpromazine (CHL). Median values of two or three independent experiments (each in triplicate) are shown. *P<0.05; **P<0.01 (D) Endocytosis of fluorescein-labeled dextran was followed under the microscope. Representative examples of DMSO and AMS36 treated cells are shown. (E) Areas and mean intensity values of the cells were measured using ZEN 2012 software and the mean intensity/1000 μm² of the cell area calculated. 28 (DMSO) and 18 (AMS36) cells were measured. Bars, 5 μm.</p

Expression of profilin 1 mutants in PC-3 cells.

<p>(A) Scheme of the DNA construct of profilin 1 mutants. (B) Restriction analysis of the DNA construct ligated into the pcDNA3 cloning vector using EcoRI and BamHI. Line 1: GeneRuler 1 kb DNA Ladder (Promega), line 2: restriction of Pfn1-Tyr139 mutant, line 3: restriction of Pfn1-Q138P mutant. (C) Western blot analysis of transfected PC-3 cell lysates expressing different profilin 1 mutants. Cell lysates were prepared 48 h post transfection. Line 1: size marker (SeeBlue® Pre-stained Protein Standard, Life Technologies), line 2: transfected empty pcDNA3 vector, line 3: transfected Pfn1-Tyr139/pcDNA3, line 4: transfected Pfn1-Q138P/pcDNA3. Mutants were detected with anti-FLAG antibodies. Detection of profilin 1 (native and mutated) and β-actin on the same membrane is also shown. Due to the chemiluminescent detection, size marker is added as a separate strip.</p

Presence of the profilin 1—clathrin complex is cathepsin X dependent.

<p>Cells were treated with cathepsin X inhibitor AMS36 (10 μM) or DMSO. Profilin 1—clathrin complexes were detected with a proximity ligation assay and analyzed with confocal microscopy. Each red dot represents a Texas red signal present on the spot with the complex. Cell nuclei were stained with DAPI. Bar, 20 μm.</p

Profilin 1 mutant Pfn1-Tyr139 decreases endocytosis and actin polymerization.

<p>(A) Cells were transfected with two plasmids carrying different profilin 1 mutants and empty plasmid (control). Clathrin-mediated endocytosis of fluorescein-labeled dextran (10 kDa) was followed with flow cytometry. Median values are representative of two independent experiments (one in triplicate and one in duplicate). *P<0.05 (B) Cells were transfected with different profilin 1 mutant constructs. After 48 hours filamentous actin was stained with phalloidin conjugate and analyzed with flow cytometry. Mean values are representative of two independent experiments (one in triplicate and one in duplicate). *P<0.05.</p

Profilin 1 as a Target for Cathepsin X Activity in Tumor Cells

<div><p>Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.</p> </div

Identification of profilin 1 as a substrate for cathepsin X carboxypeptidase activity.

<p>(<b>A</b>) Control versus AMS-36 treated sample is shown after 2D electrophoresis. The spot marked with arrow was identified as human profilin 1. (<b>B and C</b>) The C-terminal of profilin 1 (SHLRRSQY) (800 µM) was digested with recombinant cathepsin X (4.62 µM) at 37°C for 30 minutes and separated on a C18 Gemini column (5 µm, 110 Å, 150×4.6 mm) (Phenomenex). (<b>B</b>) 5 additional peaks, named peaks 2 to 6, were detected besides the original octapeptide (black line). The octapeptide control without enzyme is shown in red. (<b>C</b>) Q-TOF Premier mass spectrometry analysis of each peak showed the presence of 3 to 7 amino acid long peptides, all shortened by 1 amino acid from the C- terminal. (<b>D</b>) Profilin 1 (1 µg/µl; Abcam) was digested with recombinant cathepsin X (46.2 µM) at 37°C for several hours and the digestion product detected with mass spectrometry. A new peak was detected with molecular mass matching the mass of profilin 1 without the last amino acid residue Tyr.</p