Specific role of cytoplasmic dynein in the mechanism of action of an antitumor molecule, Amblyomin-X

Author’s Accepted Manuscript Specific role of cytoplasmic dynein in the mechanism of action of an antitumor molecule, Amblyomin-X Mario T.F. Pacheco, Kátia L.P. Morais, Carolina M. Berra, Marilene Demasi, Juliana M. Sciani, Vania G. Branco, Rosemary V. Bosch, Asif Iqbal, Ana Marisa Chudzinski-Tavassi

PII: DOI: Reference:

www.elsevier.com/locate/yexcr

S0014-4827(15)30184-1 http://dx.doi.org/10.1016/j.yexcr.2015.12.016 YEXCR10145

To appear in: Experimental Cell Research Received date: 28 August 2015 Revised date: 28 November 2015 Accepted date: 30 December 2015 Cite this article as: Mario T.F. Pacheco, Kátia L.P. Morais, Carolina M. Berra, Marilene Demasi, Juliana M. Sciani, Vania G. Branco, Rosemary V. Bosch, Asif Iqbal and Ana Marisa Chudzinski-Tavassi, Specific role of cytoplasmic dynein in the mechanism of action of an antitumor molecule, Amblyomin-X, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2015.12.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Specific role of cytoplasmic dynein in the mechanism of action of an antitumor molecule, Amblyomin-X

Mario T. F. Pacheco1*, Kátia L. P. Morais1,2*, Carolina M. Berra3, Marilene Demasi1, Juliana M. Sciani1, Vania G. Branco1, Rosemary V. Bosch1, Asif Iqbal1 and Ana Marisa ChudzinskiTavassi1**.

1

Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, Brazil

2

Department of Biochemistry, Federal University of São Paulo, São Paulo, Brazil

3

Biochemistry Department, Institute of Chemistry, University of São Paulo, São Paulo, Brazil

Running title: Dynein recruitment by Amblyomin-X

* Both authors contributed equally to this work **To whom correspondence should be addressed: Ana Marisa Chudzinski-Tavassi, Biochemistry and Biophysics Laboratory, Butantan Institute,; Av. Vital Brazil, 1500, São Paulo, SP 05503-900, Brasil; Phone: 55 11 2627 9738; Fax: 55 11 2627 9505;e-mail: [email protected].

Abbreviations: BTZ, bortezomib; FDA, Food and Drug Administration; Arf6, ADP-ribosylation factor 6; LR, lipid raft; EE, early endosomes; SE, sorting endosomes; ERC, endocytic recycling compartment; RE, recycling endosome; LE, late endosomes; Rab, Ras superfamily of monomeric G proteins, LIC2, light-intermediate chain 2 (LIC2); Rab11A, Ras-related protein Rab-11A; FIP3, protein 3 (FIP3); RE, endoplasmic reticulum; trypsin-like (T-L), PI3K, phosphoinositide-3 kinase; HRP, horseradish peroxidase; PVDF, polyvinylidene fluoride; BSA, serum albumin; TBS-T, tris-buffered saline with Tween 20; TFP, tetrafluorophenyl ester; CA, ciliobrevin A; CLP, chlorpromazine; mBCD, methyl-β-cyclodextrin; WT, wortmannin; CLQ, chloroquine; EDTA, etylenediamine tetraacetic acid; DTT, dithiothreitol, ChT-L, chymotrypsinlike; GPCR, G protein-coupled receptor.

1

ABSTRACT The Kunitz-type recombinant protein, Amblyomin-X, is an antitumor recombinant molecule from a cDNA library prepared from the salivary glands of the tick Amblyomma cajennense. The primary target of this protein appears to be the proteasome. Amblyomin-X increased gene and protein expression of distinct subunits of the molecular motor dynein, which plays a key role in the intracellular transport. Herein, Amblyomin-X was specifically taken up by tumor cells through lipid-raft endocytic pathways, but not by fibroblasts. Moreover, dynein inhibitor, ciliobrevin A, decreased Amblyomin-X uptake by tumor cells. Furthermore, incubation of tumor cells with Amblyomin-X inhibited trypsin-like activity of the proteasome, which was restored upon pretreatment with ciliobrevin A. Only in tumor cells treated with Amblyomin-X, we identified proteins bounds to dynein that are related to aggresome formation, autophagy inhibition, and early and recycling endosome markers. In addition, Amblyomin-X was found to interact with dynein, increased Rab11A protein expression and Rab11A co-localization with the light-intermediate chain 2 (LIC2) of dynein. Thereby, the results provide new insights on the antitumor mechanism of Amblyomin-X and reveal an unsuspected role of cytoplasmic dynein in its uptake, intracellular trafficking and pro-apoptotic action.

Keywords: Amblyomin-X; dynein; proteasome; plasma membrane; cancer; endocytosis, autophagy, apoptosis.

INTRODUCTION Proteasome inhibitors, such as the Food and Drug Administration (FDA)-approved drugs, bortezomib (BTZ) and carlfizomib, are commonly used to treat blood malignancies, e.g., refractory and relapsed multiple myeloma [1-3]. The proteasome inhibition induced by these molecules occurs through the chymotrypsin-like (ChT-L) activity of the proteasome [1-3]. These proteasome inhibitors are all small molecules or peptides, which are cell-permeable [1-3] and do not require specialized uptake mechanisms. Macromolecules presented in the cell microenvironment are generally taken up by endocytic mechanisms [4, 5], which may involve clathrin-dependent and independent processes [5-7]. Clathrin-independent-endocytosis requires other proteins, such as ADP-ribosylation factor 6 (Arf6), flotilin or caveolin [5-7]. In addition, endocytosis also occur through lipid raft (LR) microdomains on the plasma membrane [5-7]. LR are cell membrane regions enriched in sphingolipids and cholesterol [5-7]. The early endosomes (EE) or sorting endosomes (SE) mature in several ways to form late endosomes [6, 8]. For instance, EE/SE may merge with the plasma membrane via the fast recycling pathway; or enter the endocytic recycling compartment (ERC). In this case, the socalled recycling endosome (RE) is recycled back to the plasma membrane via the slow recycling 2

pathway. Alternatively, EE could progress into late endosomes (LE) and then into lysosomes, where endocytic cargoes are degraded [6, 8]. Selected cargoes of EE may also be transported to the trans-Golgi network [6, 8]. Endocytic trafficking is regulated by the Ras superfamily of monomeric G proteins (Rab) [6, 8] which are transported to the perinuclear region by cytoplasmic dynein [9]. For example, RE are transported via an interaction between the lightintermediate chain 2 (LIC2) of dynein and the Ras-related protein Rab11A (Rab11A) through the adapter molecule, Rab11-family interacting protein 3 (FIP3) [10]. Cytoplasmic dynein 1 is the most abundant form of dynein and is responsible for the transport of proteins and organelles towards the microtubule minus-end [11, 12]. Cytoplasmic dynein has been linked to the transport of the Golgi [13]; endoplasmic reticulum (ER) [14]; aggresomes and the autophagosomes [15]; lysosomes [11] and; endosomes [8]. Cytoplasmic dynein function in cells induced by proteasome inhibitors has been limited to aggresomes transport [1, 15], which then activates autophagy compartments, also transported by dynein [1, 2]. Amblyomin-X is a Kunitz-type recombinant protein from a cDNA library prepared from the salivary glands of the tick Amblyomma cajennense [16]. The recombinant protein showed pro-apoptotic effects in tumor cells [17-19], and decreased tumor growth and metastasis in vivo [18, 19]. The mechanism of action of Amblyomin-X involves proteasome inhibition, which occurs preferentially via the trypsin-like (T-L) activity of the proteasome [18]. Although the primary target of Amblyomin-X appears to be the proteasome [18], this macromolecule inhibits autophagy as well via a suggested mechanism involving mTOR activation, which is transported by cytoplasmic dynein [20]. Interestingly, Amblyomin-X positively modulates gene and protein expression of distinct dynein subunits, such as LIC2 [20]. The purpose of this study was to investigate whether dynein may play a role in Amblyomin-X uptake and its molecular delivery to its primary target, i.e., the proteasome, in two human tumor cell lines, such as SK-MEL-28 (melanoma) and MIA PaCa-2 (pancreatic adenocarcinoma); and in non-transformed, such as primary normal fibroblasts. In this work, we observed that dynein is essential for the Amblyomin-X's uptake and its proteasome inhibitory activity in tumor cells but not in fibroblasts. Our results indicate that the LR region of the tumor cells and phosphoinositide-3 kinase (PI3K) recruitment may be required for selective endocytosis of Amblyomin-X via a pathway not involving lysosomes. In contrast, AmblyominX is transported towards ERC by dynein hence it may be released to exert its proteasome inhibitory function. We found proteins bound to dynein after Amblyomin-X treatment that confirms the aggresome formation via the non-exclusive ubiquitin pathway [20] and reinforces the autophagy inhibition triggered by mTOR [20]. Surprisingly, our data suggested the involvement of proteins that may provide new insights on mTOR participation in Amblyomin-X's mechanism of action 3

[20] and metastasis inhibition in vivo [18, 19]. Mass spectrometry analysis of tumor cells and fibroblasts revealed distinct proteins that highlight selectivity of the tumor cells for AmblyominX upon comparison with fibroblasts, suggesting the tumor plasma membrane as a selectively tool for Amblyomin-X's uptake. Furthermore, Amblyomin-X interacted dynein and vimentin in the affinity chromatography analysis, thus confirming Amblyomin-X transport by dynein and suggesting that Amblyomin-X may be a constituent of aggresomes as well. It is noteworthy that Amblyomin-X exhibit novel effects inside the tumor cells upon comparison with proteasome inhibitors. Herein, we provide new insights into Amblyomin-X's molecular mechanism, which requires one specialized uptake mechanism, in contrast to the proteasome inhibitors. This study described for the first time, the involvement of cytoplasmic dynein in endocytosis and intracellular protein activity of a novel molecule acting on the proteasome.

MATERIALS AND METHODS Amblyomin-X preparation The recombinant molecule was obtained as described elsewhere [16].

Cell Culture The MIA PaCa-2 and SK-MEL-28 human tumor cell lines were purchased from American Type Culture Collection (ATCC) and cultured as reported elsewhere (ChudzinskiTavassi et. al., 2010; Pacheco et. al., 2014). Human primary fibroblasts were obtained as described elsewhere [18].

Antibodies The following primary antibodies were used in this work: Rab11A (Merck Millipore, Darmstadt, Germany); GAPDH (Sigma, St Louis, MO); HC1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA); LIC2 (Abcam, Cambridge, UK); secondary antibodies conjugated with horseradish peroxidase (HRP) were from Abcam, Cambridge, UK, whereas those labeled with Alexa Fluor 488 and 647 were from Invitrogen Life Technologies Inc., USA.

Western blotting analysis Protein in whole-cell lysates was quantified using a Pierce Microplate BCA Protein Assay kit (Thermo Scientific, USA). Protein expression was verified via separation in 12.5% SDS-polyacrylamide gel. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane. The samples were blocked with 5% bovine serum albumin (BSA) in tris-buffered saline with Tween 20 (TBS-T) for 1 h and then incubated with the respective primary antibodies 4

using GAPDH as an endogenous control followed by HRP secondary antibody incubation. Proteins were revealed via chemiluminescence with a homemade kit (Tris 100mM pH 8.9, pcoumaric acid 0.4 mM, luminol 2.5 mM and hydrogen peroxide 0,006% in water). Western blotting images were captured using an ImageQuant LAS 4000 (GE Healthcare, USA) and processed equally for all samples using Windows Live software for Windows7 (Microsoft, Redmond, WA, USA). No positive controls, such as proteasome inhibitors, were used in the protein expression analysis due to the lack of information regarding their action on Rab proteins expression. The analysis compared Amblyomin-X-treated with non-treated cells.

Amblyomin-X uptake Amblyomin-X was previously conjugated with a non-cell-permeable Alexa Fluor 488 using the Alexa Fluor 488 Microscale Protein Labeling kit (Invitrogen Life Technologies Inc., USA). The reactive dye contains the tetrafluorophenyl ester (TFP) portion, which is more stable in solution compared to another dyes. The conjugated protein was denominated 488Amblyomin-X (488-Amby). Cells were grown on sterile coverslips and treated with 488-Amblyomin-X alone or pretreated with the dynein inhibitor, i.e., ciliobrevin A (CA/Ambly). Culture medium was removed and then 5 µM Syto59 (Invitrogen Life Technologies Inc., USA) was added and incubated for 30 min at 37°C. Cells were washed with culture medium and analyzed with a Zeiss LSMS 510 confocal microscope (Zeiss, Germany) and LSM Image software (Zeiss, Germany). In order to investigate the uptake mechanism, cells were incubated with selected inhibitors; chlorpromazine (28 µM); methyl-β-cyclodextrin (mBCD, 28 µM); wortmannin (WT, 50 nM); or chloroquine (CLQ, 50 µM), for 30 minutes, followed by incubation with 0.5 µM 488-Amblyomin-X in the presence of inhibitors for 24 h at 37 °C. Additionally, to investigate the co-localization of 488-AmblyominX with lysosomes; the same approach was used upon incubation of cells with Lyso Tracker Red DND 99 (Invitrogen Life Technologies Inc., USA) to label lysosomes. This analysis was used to verify the uptake kinetics, lysosome co-localization and role of cytoplasmic dynein in 488-Amblyomin-X-treated cells.

Confocal Immunofluorescence microscopy Cells were grown on sterile coverslips, fixed with 4% paraformaldehyde for 15 min at room temperature, washed and permeabilized with 0.5% Triton X-100 / 0.6% etylenediamine tetraacetic acid (EDTA) 0.5 M pH 8.0 for 15 min at room temperature. The samples were blocked with 1% BSA for 30 min at room temperature and then incubated with primary 5

antibodies overnight at 4°C. The cells were then incubated with secondary antibodies for 1 h at room temperature in the dark, and the coverslips were removed from the plate and mounted in glass slides with one drop of Vecta Shield anti-fade reagent (Vecta Labs, Burlingame, CA, USA). The images were analyzed with a Zeiss LSMS 510 confocal microscope (Zeiss, Germany) and LSM Image software (Zeiss, Germany). No positive controls, such as proteasome inhibitors, were used due to the lack of information as regards their action on dynein-target interaction. The analysis compared Amblyomin-X-treated and non-treated cells. The secondary antibodies were verified to lack autofluorescence.

Proteasome activity The chromogenic substrates z-ARR-AMC and Suc-LLVY--AMC (Calbiochem, San Diego, CA, USA) were used to assess the T-L and the ChT-L activity of the proteasome, respectively, in AmblyominX-treated and non-treated cells and positive controls as described elsewhere [18].

Dynein ligands investigation The dynein HC1 chain antibody was previously coupled to magnetic beads using the Dynabeads Antibody Coupling kit (Invitrogen Life Technologies Inc., USA) and the Dynamag2 (Invitrogen Life Technologies Inc., USA). The amount of antibody used was 7 µg per 1 mg of beads. Cells were cultured in flasks, treated with compound of interest, lysed, and coimmunoprecipitated

using

Dynabeads

Co-Immunoprecipitation

kit

(Invitrogen

Life

Technologies Inc., USA), following the manufacturer’s instructions. The amount of coupled antibody-bead used was 5 mg per sample. The eluate was lyophilized and the proteins were analyzed in 10% SDS-polyacrylamide gels. The most intense bands were digested. First, the sliced bands were destained in 5% acetic acid, 50% methanol for 2 h followed by exchange of the solution and incubated for 1 h at room temperature, which have been used for all operations below. The samples were dehydrated two times with 100% acetonitrile for 5 min; reduced with dithiothreitol (DTT) 10 mM for 30 min; alkylated with iodoacetamide 50 mM for 30 min; dehydrated with 100% acetonitrile for 5 min; rehydrated with ammonium bicarbonate ((NH4)2CO3) 100 mM for 10 min; dehydrated two times with 100% acetonitrile for 5 min and evaporated in laminar flow for 5 min. Samples were digested with trypsin (20 ng/µL) in (NH4)2CO3 50 mM for 30 min on ice. The excess was removed and the samples were incubated overnight with (NH4)2CO3 50 mM. The extraction buffer 1 (5% formic acid) was added and incubated for 10 min. The extraction buffer 2 (5% formic acid, 50% acetonitrile) was added and incubated for 10 min. The samples were evaporated using the speed-vac AVC 2-18 CD Plus (Christ, Germany). The samples were 6

resuspended in extraction buffer 2 and injected into liquid chromatography mass spectrometerion trap-time of flight (LCMS-IT-TOF). The

results

were

analyzed

using

the

MASCOT

online

platform

(http://www.matrixscience.com) through the MS/MS ion search. The parameters used were: carbamidomethyl (C) as fixed modification; oxidation (M) as variable modification; peptide tolerance ± 0.5 Da; peptide charge +1, +2 and +3. No positive controls, such as proteasome inhibitors, were used due to the lack of information as regards their action on dynein. The analysis compared tumor cells and fibroblasts treated with Amblyomin-X to identify common proteins among tumor cells and differences among tumor and normal cells.

Binding of Tumor Cell Proteins to Immobilized Amblyomin-X For immobilization, A purified and lyophilized Amblyomin-X (6 mg) was dissolved in 1 ml of 0.2 M NaHCO3 containing 0.5 M NaCl, pH 8.3 and was immobilized on HiTrap™ NHS-activated HP 1 ml column (GE Healthcare Life Sciences) according to the manufacturer's instructions. After washing, the remaining active groups on the column were blocked with 0.5 M ethanolamine containing 0.5 M NaCl, pH 8.3 for 2 h at 4 °C. The electrostatically bound proteins were removed from HiTrap™ column (named HiTrap™ Amblyomin-X affinity column) by washing the four cycles with 0.2 M Tris-HCl in 0.5 M NaCl, pH 8.3, followed by 0.1 M sodium acetate in 0.5 M NaCl, pH 4.0. For negative control, the column was blocked with 0.5 M ethanolamine containing 0.5 M NaCl, pH 8.3 for overnight at 4 °C.

Tumor or non-tumor cells homogenate, typically 1 mg of homogenates were applied on HiTrap™ Amblyomin-X affinity column. To eliminate any non-specific protein interaction, the column was washed with 60 ml of 20 mM tris-HCl buffer, pH 8.3. The bound proteins were eluted with 200 mM glycine containing 0.5 M NaCl, pH 4.0. The eluent were extensively dialyzed with 25 mM ammonium bicarbonate and dried in speed vacuum evaporator (Thermo Scientific). Dried samples were stored at -80 °C or dissolved in 50 mM ammonium bicarbonate, containing 10 mM CaCl2 for MS/MS analyses. Briefly, samples were reduced with dithiothreitol (10 mM) and alkylated with iodoacetamide (55 mM) and were subsequently digested with trypsin gold (trypsin to protein ratio 1:50) in 50 mM ammonium bicarbonate, containing 10 mM CaCl2 , for 17 h at 37°C. Trypsin hydrolysates were analyzed on an EASY-nano LC system (Proxeon Biosystems) coupled online to an ESI-LTQ-OrbitrapVelos mass spectrometer (Thermo Fisher Scientific), operated in a positive mode of ionization using the data-dependent automatic (DDA) survey MS scanned tandem mass spectra acquisition. Peaks Studio 7.5 (Bioinformatics Solutions Inc. Canada) was employed for data acquisition, processing and analyses. Statistical analysis 7

Inference studies were carried out using two-way ANOVA followed by Bonferroni's post-hoc test in GraphPad Prism 5.0 software (San Diego, CA). Statistical significance was set at p ≤ 0.05.

RESULTS Amblyomin-X is endocyted by tumor cells but not by fibroblasts We first investigated whether tumor cells and fibroblasts have the ability to internalize Amblyomin-X. We labeled Amblyomin-X with cell-impermeable Alexa Fluor 488. We observed that labeled Amblyomin-X was taken up by both tumor cell lines, after 2 h of treatment, being the strongest fluorescence after 24 h of treatment (Fig. 1A). However, fibroblasts did not internalize 488-Amblyomin-X at any period of treatment (Fig. 1A). Next, we used only tumor cells to investigate the uptake mechanism, because tumor cells were able to internalize 488-Amblyomin-X. We pretreated tumor cells for 30 min with: (i) mBCD to sequester cholesterol from the plasma membrane [21]; (ii) CLP, which disassembles clathrin coats and is widely used as an inhibitor of clathrin-dependent traffic [21]; (iii) WT, an inhibitor of PI3K, which is an enzyme that regulate the phosphorylation of a large class of lipids, such as phosphatidylinositol-3-phosphate (PtdIns(3)P) and has multiple roles in membrane traffic [22]; (iv) CLQ, which abolish the acidic (H+) gradient of the EE/LE pathway [23]. We observed the absence of 488-Amblyomin-X fluorescence in either mBCD or CLQ pretreatment; a decreased 488-Amblyomin-X fluorescence in WT pretreatment; and no significant changes in CLP pretreatment; in both tumor cells (Fig. 1B).

Dynein and Rab11A participate in Amblyomin-X's endocytosis in tumor cells To explore the possible role of cytoplasmic dynein in the uptake of Amblyomin-X by tumor cells, we labeled Amblyomin-X with cell-impermeable Alexa Fluor 488 and then treated SK-MEL-28 and MIA PaCa-2 cells for 24 h with 0.5 µM of 488-Amblyomin-X. Under these experimental conditions, we observed a strong fluorescence signal concentrated in the perinuclear region, in both tumor cell lines (Fig. 2A). However, the fluorescence signal was dramatically decreased upon pretreatment of cells with ciliobrevin A (CA) (Fig. 2A). Next, we examined the protein levels of Rab11A and its co-localization with dynein, because Rab proteins are involved in endosome trafficking [7, 8], and it is a binding-partner of LIC2 [10]. Both tumor cell lines treated with Amblyomin-X showed increased Rab11A after 4 h and 24 h (Fig. 2B and 2C) in contrast to the fibroblasts that did not show any changes (Fig. 2D). Moreover, Amblyomin-X increased Rab11A after 2 h in MIA PaCa-2 cells (Fig. 2C). We used fibroblasts in the experiments involving dynein and Rab11A in order to confirm that

8

endocytosis did not occur. Our next step was to investigate co-localization of LIC2 and Rab11A. We observed an increased co-localization of LIC2 and Rab11A in both tumor cells after 24 h of Amblyomin-X treatment but not in fibroblasts (Fig. 2E). We also examined whether 488-Amblyomin-X co-localized or not with lysosomes. In this experiment, we excluded fibroblasts as no endocytosis was observed (Fig. 1A and 2E). We observed that 488Amblyomin-X did not co-localize with lysosomes in both tumor cells after 24 h of treatment (Fig. 2F).

Dynein inhibition affects the Amblyomin-X's action on the proteasome of tumor cells Next, we investigated whether recruitment of dynein is required for the proteasome inhibition induced by Amblyomin-X [18]. We have previously shown that Amblyomin-X preferentially inhibits the T-L activity of the proteasome in SK-MEL-28 and MIA PaCa-2 cells after 4 h (using 1 µM), or after 24 h (using 0.5 µM) of incubation [18]. Here, we used the lowest concentration of Amblyomin-X sufficient to detect proteasome inhibition. Amblyomin-X decreased the T-L activity of the proteasome in both tumor cells after 24 h of treatment, as opposed to BTZ and MG-132 (Fig. 3A and 3B). However, Amblyomin-X did not induce any changes in fibroblasts (Fig. 3C). Interestingly, CA treatment alone or CA pretreatment (CA/Ambly) induced no changes in the T-L activity of the proteasome in tumor cells and fibroblasts (Fig. 3A, 3B and 3C). In contrast, Amblyomin-X did not induce any changes in the ChT-L activity of the proteasome in tumor cells and fibroblasts (Fig. 3A, 3B and 3C). Surprisingly, CA treatment alone decreased the ChT-L activity of the proteasome in both tumor cells, as BTZ and MG-132 treatment did (Fig. 3A and 3B). Although, in fibroblasts, CA treatment alone showed a slight decrease of the ChT-L activity, it was not statistically significant (Fig. 3C). However, Amblyomin-X post treatment (CA/Ambly) induced no changes in the ChT-L activity when comparing CA/Ambly treatment and negative control (treated with vehicle) in tumor cells and fibroblasts (Fig. 3A, 3B and 3C). Indeed, CA/Ambly treatment increased the ChT-L activity of the proteasome upon comparison among CA/Ambly treatment and CA treatment alone in tumor cells as well as in fibroblasts (Fig. 3A, 3B and 3C).

Receptors, signal transducers and transcription factors were bound to dynein after Amblyomin-X treatment in tumor cells To investigate the dynein cargo content, we analyzed the protein profile of dynein ligands after Amblyomin-X treatment, in SK-MEL-28 and MIA PaCa-2 cells and fibroblasts. We targeted the motor subunit of cytoplasmic dynein, HC1, which has a molecular mass of 500 kDa, approximately. We found a similar protein band profile among the tumor cells, after Co-IP 9

followed by separation in SDS-polyacrylamide gel (Fig. 4A). Further, mass spectrometry analysis showed that Amblyomin-X treatment induced the interaction of diverse proteins with dynein (Fig. 4B). The proteins in common, among the tumor cells, were classified on the basis of their biological function and were subdivided into the following groups: apoptosis (5); cell cycle (2); cytoskeleton / extracellular matrix (ECM) / adhesion (10); proteolysis (1); metabolism (6); transport / endocytosis (2); proteasome (2); aggresome (2); protein synthesis (2); RNA processing (2); autophagy (1); signal transduction (8); transcription factor (3); receptor (3); polarity (1); chromosome / nucleosome (1) and; unknown function (1) (Fig. 4B). The proteins, which bound to dynein in both tumor cells, after Amblyomin-X treatment, were: Rab20; H1e; BACE2; sIL-6R; vimentin, CasZ1; SNIP; synaptopodin; TRAF4; Rab11A; Dsel; SHMT1; FBXL13; NDUFV1; ME2GLYDH; ZDHHC14; TMEM26; TNFR; Bcl-2; PCCase-β; RhoGEF18; CaRF; Misu; LysRS; ADAMTS-3; nibrin; EGFL6; N-CoR2; cGK2; integrin-β8; PKC-α; Rpn13; Rpn10; PARD3; laminin-1α;

Bag3 and; Raptor (Table 1S).

However, Amblyomin-X was not found in the mass spectrometry analysis. Interestingly, the protein band profile in fibroblasts showed just one clear band common with both tumor cells (Fig. 4A). Only two proteins were found and they were distinct from the tumor cells. The proteins, which were bound to dynein, after Amblyomin-X treatment, were PNPLA1 (metabolism) and ZNF354C (transcription factor) (Table 2S). The biological function of each protein described above and listed in Table 1S and 2S were obtained from Uniprot (http://www.uniprot.org). Furthermore, the two proteins that bound to dynein in fibroblasts (Table 2S) present a molecular weight that are in the range of 50 - 70 kDa. In this context, the proteins that show the same molecular weight range, in both tumor cells, were BACE2, Vimentin, TRAF4, SHMT1, NDUFV1, ZDHHC14, PCCase-β and EGFL6 (Table 1S). The proteins found at this range comparing the tumor cells and fibroblasts are shown in the Fig. 4A. Binding of cytoplasmic Dynein to Immobilized Amblyomin-X Finally to check whether Amblyomin-X interact with dynein, the homogenates of the tumor cell lines i.e. MIA PaCa-2 and SK-MEL-28 were applied to immobilized Amblyomin-X column. Several proteins including dynein heavy chain were identified in eluent fraction that bound to immobilized Amblyomin-X (data not shown). The list of some interesting proteins that correlates with the immunoprecipitation experiment, including number of peptides and peptides unique identified in eluent fraction that bound to immobilized Amblyomin-X is given in table S3. It includes proteins such as, dynein, H1e, SHMT1 and vimentin. No such proteins were identified in negative control. Fibroblasts was not used in this experiment because AmblyominX was not internalized in this cell, thus we aimed in experiments that may explain the effects of Amblyomin-X presence in the cell microenvironment as previously demonstrated [20].

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DISCUSSION This study described the mechanism of action of a Kunitz-type recombinant protein, acting on the proteasome, regarding a new molecular target, i.e. dynein. Amblyomin-X is a protein [16] not a small molecule or peptide, such as the proteasome inhibitors described [1-3]. As the proteasome inhibitors pass through the plasma membrane [1-3] without requiring any specialized uptake mechanism, we investigated Amblyomin-X internalization by the tumor cells, SK-MEL-28 and MIA PaCa-2; and a non-tumor or normal cell, i.e., fibroblasts. We described that Amblyomin-X regulated gene expression of dynein LIC2 chain [20] and other dynein chains [20]. We also showed that the recombinant protein induced protein overexpression of distinct dynein chains, especially LIC2, after Amblyomin-X treatment [20]. Furthermore, this molecule induced aggresome formation, increased K63 polyubiquitinated proteins, autophagy inhibition with suggested mTOR activation and dynein assistance in transporting aggresomes and mTOR [20]. As K63 signaling may be related to the endosome formation [15]; dynein transports organelles and vesicles, such as endosomes [9]; and considering that Amblyomin-X is a protein [16]; we analyzed the probable endosome formation involving dynein. We found that labeled Amblyomin-X was internalized by both tumor cells, but this effect was abolished upon dynein inhibition using CA pretreatment. In addition, when the cells were pretreated with endosomal inhibitors; the results suggested the endocytosis via the LR microdomain, because the cholesterol removal by mBCD abolished the fluorescence of 488Amblyomin-X and LR's are rich in cholesterol [4, 5, 7]. The results also suggested the participation of the PI3K in Amblyomin-X's endocytosis, because the pretreatment with WT decreased 488-Amblyomin-X fluorescence and PI3K is involved in membrane trafficking [22]. Interestingly, dynein was identified in homogenates fraction of both tumor cells that bound to immobilized Amblyomin-X (Table 3S). We identified several other proteins bound to Amblyomin-X that were common to the proteins identified by immunoprecipitation. These proteins either may interacted directly with Amblyomin-X or co-eluted with dynein in a protein complex. Moreover, vimentin was also bound to Amblyomin-X, thus suggesting that the recombinant protein may be a constituent of aggresomes transported to dynein. In this context, it is possible that Amblyomin-X may be destined to proteolysis by the proteasome and then aggregates in aggresomes vesicles. Is Amblyomin-X inhibiting the proteasome by steric blockage? Is Amblyomin-X partially processed by the proteasome and then inhibits it in a steric way or chemical reaction? Why trypsin-like activity of the proteasome is preferentially inhibited by Amblyomin-X? This study revealed novel hypothesis that requires further investigation. Nonetheless, it strongly support our perception that dynein play important role in the internalization of Amblyomin-X by tumor cells.

11

Amblyomin-X induced Rab11A overexpression and its co-localization with LIC2. We found either Rab11A or Rab20 bound to dynein after Amblyomin-X treatment. The Rab proteins are strictly related to the regulation of membrane trafficking [6, 8]. Furthermore, Rab20 is a marker for EEs whilst Rab11A is a marker for REs [6, 8]. In addition, Rab11A is a bindingpartner of LIC2 [10]. Additionally, Amblyomin-X did not co-localize with lysosomes. The labeled Amblyomin-X was found in the perinuclear region where the ERC is present [6, 8]. Therefore, it is possible to suggest that the recycling endosomes could be an Amblyomin-X intracellular destination in tumor cells. This hypothesis is enhanced when considering that no increase in the acidic vesicles formation was observed upon prolonged treatment of tumor cells with Amblyomin-X [20], and that no LE markers were found in the proteome analysis of dynein ligands in both tumor cells. The current hypothesis is that a full-length Amblyomin-X molecule may be released in the ERC because RE do not present a high enzyme content as LE and lysosomes do [24-26]; or Amblyomin-X may be somehow cleaved in the plasma membrane. Surprisingly, 488-Amblyomin-X was not internalized in fibroblasts. In this cell type, Amblyomin-X did not regulate Rab11A protein expression or co-localized dynein (LIC2) and Rab11A. In other words, these molecules involved in endosome transport are not required in fibroblast. It suggests a possible lack of a membrane receptor or other molecule anchor that may alter the Amblyomin-X entrance. However, further investigation is necessary regarding its selectivity for the tumor cells and not for fibroblasts. Furthermore, the proteasome-aggresomeautophagy pathway, dynein gene, protein regulation and transport studies in fibroblasts are required; as we did with the tumor cells[20], although our group showed that Amblyomin-X was not cytotoxic for fibroblasts [18]. Amblyomin-X primarily inhibits the T-L activity of the proteasome [18]. We found that the molecule decreased the T-L activity in both tumor cells that was abolished when the cells were pretreated with CA. Interestingly, CA treatment alone decreased the ChT-L activity and the CA/Ambly treatment increased the same parameter upon comparison between CA and CA/Ambly treatment. CA is a small molecule inhibitor of the ATPase activity of dynein and of the Hedgehog pathway [27]. The proteasome also present ATPase activity [28]. Moreover, the ChT-L is usually the first activity triggered by the proteasome to degrade the protein substrate [28]. Thereafter, the CA treatment alone may possibly inhibited the ChT-L activity of the proteasome. We conclude that dynein function is required for the inhibitory activity exerted in the proteasome by Amblyomin-X in both tumor cells. However, Amblyomin-X did not require dynein for the proteasome inhibition in fibroblasts, because the proteasome was not inhibited. It is noteworthy that CA/Ambly treatment increased the ChT-L activity in fibroblasts when compared to CA alone. This result may be explained by increased cell viability (data not shown) and not for the absence of the 12

Amblyomin-X action regarding previously dynein inhibition, because 488-Amblyomin-X was not internalized and the proteasome was not inhibited in fibroblasts. Proteomic analysis, in both tumor cells indicated that the dynein ligands were mostly bound to this molecular motor via endocytic (Rab11A, Rab20) or aggresomes

vesicles

(vimentin, Bag 3, proteasome components of the regulatory 19S particle: Rpn10 and Rpn13), because after 24 h of Amblyomin-X treatment, this molecule was taken up by tumor cells; the proteasome is inhibited [18] and; the aggresomes were formed [20]. In addition, some of the proteins found may bound via direct interaction between dynein and the target, because dynein transports proteins, such as the PARD3 [29] and transcription factors, such as the nuclear factor kappa B (NF-κB) [30]. Indeed, PARD3 was found in the proteome analysis, supporting the hypothesis that PARD3 may possibly interact directly with LIC2, because LIC2 is a binding-partner of PARD3 [29], and Amblyomin-X increased the gene and the protein expression of LIC2 [20]. Thus, LIC2 appears to play specific roles in the mechanism of action of Amblyomin-X, such as endosome transport via Rab11 interaction beyond the PARD3 interaction. The PARD3 protein is associated to cell polarity and may be found in the tight junction of the cells and in PKC-containing complexes [31]. Interestingly, PKC-α was bound to dynein in both tumor cells treated with Amblyomin-X. Surprisingly, CasZ1 is a transcription factor involved in cell adhesion, vascular assembly and morphogenesis [32]. In summary, intracellular pathways and structures that contribute to cell adhesion and migration, such as tight junctions [33]; gap junctions [31]; focal adhesion junctions [34] and; ECM components [35, 36] may play important roles in the mechanism of action of Amblyomin-X, because ten proteins related to cytoskeleton / ECM / adhesion were linked to dynein, i.e.: vimentin, synaptopodin, ADAMTS3, integrin-β8, laminin-1α, SNIP, EGFL6, PKC-α, PARD3 and RhoGEF18. These findings may suggest pathways related to the metastasis reduction observed in vivo, that were induced by Amblyomin-X [18, 19], although this hypothesis requires further investigation. Our group demonstrated that Amblyomin-X induced mitochondrial dysfunction [37]. We found proteins related to the cell metabolism and cell respiratory chain, bound to dynein after treatment with the recombinant protein in both tumor cells. The proteins found in the dynein proteome analysis were: Dsel, SHMT1, NDUFV1, ME2GLYDH, ZDHHC14 and PCCase-β. Amblyomin-X also bound a protein involved in metabolism, i.e, SHMT1. We hypothesize that the other proteins may be primarily linked to dynein via the aggresome structures in response to the mitochondrial impairment induced by Amblyomin-X [37]. Moreover, the histone H1e involved in nucleosome assembly [38], was linked to dynein in both tumor cells. It was also found bound to immobilized Amblyomin-X (Table 3S). We know that Amblyomin-X induces DNA damage [18]. Therefore, our hypothesis is that this histone is essential for both tumor cells in the structure of the genetic cell cargo and that 13

Amblyomin-X may sequester H1e, thus impairing the nucleossome assembly. This event my affect and contribute to the DNA damage induced by Amblyomin-X in the tumor cells studied [18]. Herein, we found three receptors bound to dynein in the tumor cells treated with Amblyomin-X, i.e., integrin-β8, TNFR and sIL-6R. Amblyomin-X was able to bound integrinβ8 in SK-MEL-28 cell line as well. Therefore, this interaction may be related to the antimetastatic activity of Amblyomin-X in vivo [19] which is characteristic of melanoma tumors, but this requires further investigation. The endocytic vesicles may trigger intracellular pathways via activation of receptors that were internalized together with the macromolecule [4, 5]. Hence, the results indicated that TNFR may triggered an intracellular pathway mediated by TRAF4, because this adapter molecule was bound to dynein and TRAF4 is a signal transducer of TNFR [39]. In addition, the soluble form (sIL-6R) of the combined interleukin-6 receptor (IL-6R) and glycoprotein 130 (gp130) via trans-signaling [40, 41], was bound to dynein after Amblyomin-X treatment in both tumor cells. Furthermore, the formation of sIL-6R triggers the JAK / STAT pathway [41], involved in cell proliferation, cell migration and apoptosis [41]. It is noteworthy that intracellular pathway described above requires further investigation. The recombinant protein as well induced the inhibition of autophagy, that was suggested to occur via mTOR activation assisted by dynein transportation, in the tumor cells studied [20]. The mTOR complex responsible for autophagy activation is the mTORC1 containing the mTOR kinase and other proteins that distinguishes this complex from the mTORC2 complex, such as the Raptor protein [42, 43]. We found Raptor bound to dynein after Amblyomin-X induction in both tumor cells, thus supporting the autophagy inhibition by the mTORC1 complex and its transportation by dynein. Thus, the mTOR may be activated by an intracellular pathway. The PI3K / protein kinase B (Akt) pathway is a well-known mechanism that activates mTOR complex [43]. However, the PI3K / Akt pathway may be triggered by TNFR as well [44], or may exhibit a crosstalk with JAK / STAT pathway [40], or Smad pathway [45, 46]. Therefore, either TNFR or sIL-6R bound to dynein may possibly activated PI3K / Akt pathway directly or indirectly in response to Amblyomin-X treatment. In addition, corroborating with all findings previously discussed, the hypothesis of PI3K / Akt / mTOR pathway activation in the tumor cells studied is reinforced by the Amblyomin-X's internalization assays. When both tumor cells were pretreated with WT, the fluorescence signal was decreased; thus suggesting that PI3K may be not crucial for Amblyomin-X's endocytosis but may assist in this cell event. As the endosome formation may trigger distinct intracellular pathways [4, 5], the PI3K may be possibly being recruited to the tumor plasma membrane and activated during endocytosis. 14

Our group has demonstrated the anti-coagulant activity of Amblyomin-X [19]. In this context, we found the protein RhoGEF18 bound to dynein in both tumor cells, that is activated by the Gβγ subunit of the heterotrimeric G protein (Gαβγ) [39, 47]. Therefore, the possibly activation of RhoGEF18 may occur upon activation of Gαβγ linked to a G protein-coupled receptor (GPCR). Thereby, the involvement of the GPCR related to coagulation and cancer may be a feasible hypothesis to be investigated in the future, for example, protease-activated receptor 2 (PAR2), that is a GPCR [48]. Surprisingly, only two proteins were found bound to dynein in fibroblasts, after Amblyomin-X treatment in the same range of 50 to 70 kDa of the tumor cells studied. However, the two proteins were different from those found bound to dynein in the tumor cells. The first one is a transcriptional repressor, i.e., ZNF354C [49], that may be related to a probable Amblyomin-X's disturbance due to the recombinant protein presence in the cell microenvironment. However, this hypothesis requires further investigation, because Amblyomin-X did not decrease fibroblast's viability [18]. The second protein found was PNPLA1, which is a protein possessing a phospholipase domain acting on the phospholipid catabolism (also termed as glycerophosholipid) [50]. In this context, the results suggested a membrane compartment in which fibroblasts did not internalize Amblyomin-X. Would the PNPLA1 protein found indicate that Amblyomin-X uptake involves a phospholipid catabolism reaction mediated by proteins with phospholipase domains and transportation by dynein? And that the phospholipid catabolism was somehow blocked in the presence of Amblyomin-X in the fibroblast microenvironment? Are there other normal cells that do not undergo endocytosis upon Amblyomin-X treatment? These questions requires further investigation and may provide a valuable data to study tumor cell selectivity. In this study, we investigated the role of dynein in the uptake mechanism of Amblyomin-X and its transport towards the proteasome, as well in the pro-apoptotic effects (proteins in the immunoprecipitate PARD3, mTOR); which suggests that endocytosis may be the entrance route of the recombinant protein inside the tumor cells. Moreover, dynein appears to be a novel molecular target of Amblyomin-X. The results showed that this novel molecule acting on the proteasome requires a specialized uptake mechanism assisted by dynein, as opposite to proteasome inhibitors described [1-3]. Furthermore, dynein exhibits a remarkable function in transporting endosomes and proteins in both tumor cells. These functions relies on the transportation of diverse components found in either endosome or aggresome structures; or even via directly interaction with proteins and transcription factors in the tumor cells studied. This data distinguishes Amblyomin-X from the proteasome inhibitors, which requires dynein function only for aggresome and autophagic components transport [1-3, 15]. Moreover, Amblyomin-X do not trigger autophagy in both tumor cells [20] as the proteasome inhibitors do [1, 2]. 15

Hence, the results require more experimental data to confirm tumor cell selectivity as well as the possible intracellular pathways in tumor cells and the mechanism by which fibroblasts did not internalize Amblyomin-X. However, the study revealed Amblyomin-X routes and its destination to the ERC and possibly the aggresomes, which may be correlated to the proteasome inhibition after ERC release the recombinant protein. Amblyomin-X is, therefore, a promising molecule to treat malignant tumors and exhibits distinct features from the classic proteasome inhibitors.

Acknowledgments:

We gratefully acknowledge Dr. Roger Chammas and Dr. Renata F.

Saito from the Cellular Biology laboratory from ICESP for their assistance with the western blotting analysis

and

their

helpful

discussions.

We

thank

the

Parasitology and

Immunopathology laboratory from the Butantan Institute for their assistance with confocal experiments. We also acknowledge Prof. Giampietro Schiavo from Sobell Department of Motor Neuroscience & Movement Disorders, UCL Institute of Neurology University, College London for discussions and critical reading of the manuscript. We also thank for the financial support provided by the São Paulo Research Foundation (FAPESP; processes 2000/11624-5, 2011/05969-4, 2010/07958-7 and CETICs 2013/07467-1)

Conflict-of-interest disclosure: The authors declare that they have no conflicts of interest with the contents of this article.

Author Contributions: Conceived and designed experiments: MTFP, KLPM, CMB, MD, AMCT. Performed the experiments: MTFP, KLPM, AI. Analyzed the data: MTFP, KLPM. Contributed reagents/materials/analysis tools: JMS, VGB. Wrote the paper: MTFP. Revised the paper: KLPM, AMC-T, AI. Coordinated the study: AMC-T.

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FIGURE 1. Amblyomin-x's uptake kinetics and mechanism. (A) Confocal microscopy analysis of live cells (SK-MEL-28, MIA PaCa-2 and fibroblasts) treated with 0.5 µM 488-Amblyomin-X for 5 min, 30 min, 2h, 4h and 24 h. The final overlay image represents five fields of three independent experiments in which the green fluorescence represents labeled Amblyomin-X (488-Amblyomin-X), while the red fluorescence represents the nucleus stained with 5 µM 19

Syto59. (B) Confocal microscopy analysis of live cells (SK-MEL-28 and MIA PaCa-2) treated with 0.5 µM 488-Amblyomin-X for 24 h; or 28 µM CLP for 30 min; or 28 µM mBCD for 30 min; or 50 µM WT for 30 min; or 50 µM CLQ for 30 min. The final overlay image represents five fields of three independent experiments in which the green fluorescence represents labeled Amblyomin-X (488-Amblyomin-X), while the red fluorescence represents the nucleus stained with 5 µM Syto59. FIGURE 2. Endosome transport by dynein. (A) Confocal microscopy analysis of live cells (SKMEL-28 and MIA PaCa-2) treated with 0.5 µM 488-Amblyomin-X for 24 h; or 100 µM CA for 24 h followed by 0.5 µM 488-Amblyomin-X for 24 h. The final overlay image represents five fields of three independent experiments in which the green fluorescence represents labeled Amblyomin-X (488-Amblyomin-X), while the red fluorescence represents the nucleus stained with 5 µM Syto59. Representative western blots of whole-cell lysates of Rab11A in (B) SKMEL-28; (C) MIA PaCa-2 or (D) fibroblasts; of cultured cells treated with vehicle (PBS) or 0.5 µM Amblyomin-X for 2 h, 4 h or 24 h. Images are representative of three independent experiments. (E) Confocal microscopy analysis of fixed cells (SK-MEL-28, MIA PaCa-2 and fibroblasts) treated with vehicle (PBS) or 0.5 µM Amblyomin-X for 24 h. The final overlay image represents five fields of three independent experiments in which the green fluorescence represents Rab11A, while the red fluorescence represents LIC2 chain of dynein. The yellow fluorescence represents co-localization in the final merge image. (F) Confocal microscopy analysis of live cells (SK-MEL-28 and MIA PaCa-2) treated with 0.5 µM 488-Amblyomin-X for 24 h. The figures represents five fields of three independent experiments in which the green fluorescence represents labeled Amblyomin-X (488-Amblyomin-X), while the red fluorescence represents the lysosomes stained with 50 nM Lyso Tracker Red DND 99. FIGURE 3. Dynein role in the proteasome inhibition induced by Amblyomin-X. Cultured cells (SK-MEL-28, MIA PaCa-2 and fibroblasts) were treated with vehicle (PBS), 0.5 µM Amblyomin-X for 24 h, 5 µM MG-132 for 24 h, 100 nM bortezomib for 24 h, 100 µM CA for 24 h or 100 µM CA for 24 h followed by 0.5 µM Amblyomin-X for 24 h. The T-L and ChT-L activity was assessed with chromogenic substrates using whole-cell lysates of (A) SK-MEL-28 cells; (B) MIA PaCa-2 cells or (C) fibroblasts. Results are reported as the means ± standard error of three independent experiments. The criteria and representation of statistical significance were set as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 or ns (non-significant). FIGURE 4. Dynein ligands profile in tumor cells and fibroblasts stimulated with Amblyomin-X. (A) Cultured cells (SK-MEL-28, MIA PaCa-2 and fibroblasts) were treated with 0.5 µM Ambly for 24 h. Whole-cell lysates were incubated with dynein antibody-containing-magnetic beads and the eluted fraction was separated via electrophoresis. The gels were stained with Coomassie blue to visualize the protein band profile of dynein ligands. Image is representative of three independent experiments. The red arrowhead indicates the common highest protein band in both tumor cells whilst the blue arrowhead indicates the protein band found in fibroblasts with the same molecular weight range of the most intense band of the tumor cells. (B) Mass spectrometry analysis of the most intense bands sliced from the gel derived from Co-IP in common with both tumor cells (SK-MEL-28 and MIA PaCa-2). The graphic represents the number of proteins in common between both tumor cells (SK-MEL-28 and MIA PaCa-2), separated by its primary biological function. The list of proteins can be found in Table 1S (tumor cells) and 2S (fibroblasts), respectively. Note that only two proteins were found in fibroblasts and are described in Table 2S; therefore there was no graphic bars.

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Graphical abstract 21

Highlights 1. Amblyomin-X acts preferentially in tumor cells by proteasome inhibition. 2. Different of others proteasome inhibitor, Amblyomin-X requires activating endocytosis. 3. Dynein play a role in the Amblyomin-X mechanism of action.

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