UBE2O ubiquitinates PTRF/CAVIN1 and inhibits the secretion of exosome-related PTRF/CAVIN1

Plasmids

Both full-length human PTRF and SDPR from cDNA of HeLa cells were cloned into pCR3.1-Myc and lentivirus vector pLV-Flag. Plasmids including pCR3.1-UBE2O-Myc, pCR3.1-UBE2O-C1040S-Myc, His-HA-Ubi, His-HA-Ubi-KO and pLV-Flag-UBE2O had been described in the previous studies [30, 40]. UBE2O deletion constructs (D1–D5), PTRF deletion constructs (D1–D3) and SDPR deletion constructs (D1–D2) were cloned into pLV-Flag vector. PTRF deletion constructs D3 was also cloned into pGEX-5X-1 vector. And the truncated version of the conserved region 2 (CR2) of UBE2O was cloned into pET28A vector. All the primers for UBE2O deletion constructs D1–D5, PTRF deletion constructs D1–D3, SDPR deletion constructs D1–D2 and the truncated version of UBE2O CR2 were shown in Additional file 1: Table S1. With regard to the construction of vectors for PTRF and SDPR knockdown, PTRF or SDPR shRNA forward primer and reverse primer were complementary paired and followed by inserted into an inducible shRNA lentivirus vector. The primers for four PTRF shRNA sequences and three SDPR shRNA sequences also can be found in Additional file 1: Table S1. All plasmids were verified by DNA sequencing.

Cell culture, transfection and stable cell lines construction

HEK293T and HeLa cells, obtained from ATCC, were cultured in DMEM/High glucose (HyClone, SH30022.01) supplemented with 10% fetal bovine serum (FBS, Gibco, 10270-106) and 100 U/ml penicillin/streptomycin (HyClone, SV30010). Cells were cultured in the condition of 37 °C and 5% CO2, and had been tested for mycoplasma (Lonza, LT07-318).

Plasmids were transfected into cells by transfection reagent poly-ethylenimine 25 K (PEI 25 K, Polysciences, Inc, 23966-100). After 36 h transfection, cells were collected for analysis by immunoprecipitation assays or ubiquitination assays.

Lentiviruses were produced by transfecting virus packaging plasmids pRSV-REV, pCMV-VSVG and pMDLg-RRE (gag/pol) with the desired plasmid into HEK293T. Cell supernatants collected after 48 h transfection were used to infect HeLa cells with 8 μg/ml of polybrene (Sigma). Cells were selected by 200 μg/ml hygromycin B (Sigma, V900372-250MG) or 1 μg/ml puromycin (Sigma, P8833-10MG) after at least 2 days infection. For UBE2O knockout cell lines, UBE2O sgRNA forward primer caccgcatctatcccgtcaacagca and UBE2O sgRNA reverse primer aaactgctgttgacgggatagatgc were cloned into pSpCas9(BB)-2A-GFP [41]. Cells transfected with sgRNA expression plasmid were sorted by fluorescence-activated cell sorter (MoFlo Astrios, USA) 48 h after transfection. Single cells were re-seeded and cultured with the normal medium in the presence of 1/1000 TG/BCS (0.433% α-Thioglycerol (α-TG, Sigma, M6145), 20 μM bathocuproinedisulfonic acid disodium salt (BCS, Sigma, B1125)). UBE2O knockout cell lines were verified by western blot and DNA sequencing.

Immunoprecipitation assays

For immunoprecipitation assays, cells were lysed with WCE lysis buffer (10% glycerol, 150 mM NaCl, 50 mM Tris–HCl, pH 8.0, 0.5%NP40) plus 1 × protease inhibitor cocktail (Bimake, B14001) on ice for 20 min. Cell lysates were centrifuged at 14,000 rpm, 4 °C for 10 min. 50 μl supernatant was aliquoted as input, and Flag M2 beads were used for immunoprecipitation (Sigma, M8823) at 4 °C for 1.5 h. For endogenous immunoprecipitation assays, cell lysates were incubated with PTRF antibody (Proteintech, 18892-1-AP) or UBE2O (Bethyl Laboratories, A301-873A) overnight. Then, the cell lysates with PTRF antibody were incubated with protein A resin (GenScript, L00210) for 2 h, and the cell lysates with UBE2O antibody were incubated with protein A/G magnetic beads (MedChemExpress, HY-K0202) for 2 h. After the incubation, beads which included Flag, protein A or protein A/G beads were washed by WCE lysis buffer for three times. Both the input samples and the immunoprecipitated samples were boiled with 2 × SDS loading buffer at 95 °C for 10 min. All the boiled samples were analyzed by western blot.

Ubiquitination assays

Cells were washed twice with cold phosphate-buffered saline (PBS, BI, 02-024-1ACS) with 10 mM N-Ethylmaleimide (NEM, Sigma, E3876-5G) and lysed with 8 M urea buffer solution (8 M urea, 10 mM imidazole, 10 mM Tris–HCl pH 8.0, 0.1 M NaH2PO4, 0.1 M Na2HPO4) with 10 mM β-mercaptoethanol (Macklin, M828395-100 ml). Lysates were centrifuged at 14,000 rpm, room temperature for 10 min. The supernatant was incubated with nickel resins (GenScript, L00223I-50) at room temperature for 2 h. Nickel beads were washed 3 times by 8 M urea buffer solution with 20 mM imidazole, and protein was eluted with 2 × SDS loading buffer.

Purification of PTRF-D3 and UBE2O-CR2

Protein expression was performed using BL21 strain of E. coli (Vazyme, C504-02) with plasmids of pGEX-5X-1, pGEX-5X-1-PTRF-D3 and pET28A-UBE2O-CR2. GST protein was induced at 37 °C with 0.25 mM isopropyl thiogalactoside (IPTG) for 6 h, GST-PTRF-D3 was induced at 25 °C with 0.25 mM IPTG overnight, and His-UBE2O-CR2 was induced at 37 °C with 0.25 Mm IPTG for 6 h. Bacteria were collected at 4000 rpm, 4 °C for 15 min and were re-suspend by PBS, lysozyme (Macklin, L6051-25 g) and benzonase nuclease (Sigma, E1014-25KU). Follow by ultrasound pyrolysis, samples were centrifuged at 14,000 rpm, 4 °C for 10 min. The supernatant was filtered by the 0.45 μm filter (Millipore, SLHV033RB). Lysates with GST or GST-PTRF-D3 protein were incubated with glutathione resin (GenScript, L00206-100) and eluted by 10 mM L-glutathione (Biofroxx, YS-1392GR005), while lysates of His-UBE2O-CR2 protein were incubated with nickel resins and eluted by 50 mM imidazole eluent (50 mM Tris–HCl, pH 8.0, 0.15 M NaCl, 50 mM imidazole). All eluted proteins were finally concentrated by the 10 K centrifugal filter (Millipore, UFC801008).

In vitro interaction

His-UBE2O CR2 protein was incubated with nickel resins at 4 °C for 1.5 h. Followed by three washed with WCE lysis buffer, GST or GST-PTRF-D3 protein was added to immunoprecipitates and was incubated at 4 °C overnight. Nickel beads were washed with WCE lysis buffer for three times and boiled with 2 × SDS loading buffer for western blot.

Exosomes isolation

Approximately 5 × 106 cells were seeded in cell culture dishes. To make the data more accurate, the same number of cells were seeded in different comparison groups each time. Then, cells were cultured in normal medium until 80% confluent, followed by 3 PBS washes and 18 mL of serum-free medium culturing for an additional 40 h. Next, the serum-free conditioned medium was harvested and centrifuged sequentially at 300 × g and 2000 × g for 10 min to remove cells and cell debris, respectively. Small cell debris and larger microvesicles were removed by 10,000 × g centrifugation for 30 min (Avanti J-E, Beckman, USA). Finally, the exosome-rich pellet was collected after ultracentrifugation at 167,000 × g for 70 min (XPN-100, Beckman, USA). The exosome-rich pellet was washed by PBS and ultracentrifuged at 167,000 × g for 70 min again [42, 43]. Exosomes were aliquoted for the following experiments.

Western blot and BCA assay

For the quantification of total protein content in exosomes, exosomes and their host cells were collected and were lysed by RIPA lysis buffer (Beyotime, P0013K). To ensure exosomes were secreted by the similar number of cells in the different comparison group, the detection amount of exosome lysates was adjusted according to the total protein content of their host cells. The total protein content of exosomes was quantified by the Pierce BCA protein assay kit (Thermo, 23227).

For western blot, protein concentrations of cell lysates were also quantified by the Pierce BCA protein assay kit according to product instructions. Equal amounts of protein samples were boiled in 1 × SDS loading buffer and resolved by SDS-PAGE. Proteins were transferred to the PVDF membrane (Millipore, ISEQ00010) at 50 mA overnight (Bio-Rad, USA). Membrane was incubated in blocking buffer (5% skim milk powder (Biofroxx, 1172GR500) in 1 × TBST, 50 mM Tris–Cl, pH 7.6, 150 mM NaCl, 0.1% Tween 20) for 1 h. Then, the membrane was incubated in the primary antibodies for 2 h and in the secondary antibody for 1 h at room temperature. Western blot was visualized using ChemiDoc Imaging systems (Bio-Rad, USA). Blots were quantified by ImageJ.

Antibodies used in this study included PTRF (Proteintech, 18892-1-AP), SDPR (Proteintech, 12339-1-AP), Flag M2 (Sigma-Aldrich, F1804), Flag (Sigma-Aldrich, F7425-2MG), GST (Invitrogen, PA1-982A), UBE2O (Invitrogen, PA5-54839), UBE2O (Bethyl Laboratories, A301-873A), HA (Sigma-Aldrich, H3663-200UL), Myc (Proteintech, 16286-1-AP), His (Santa Cruz, sc-8036), TSG101 (Abcam, ab125011), CD63 (Proteintech, 25682-1-AP), CD9 (Proteintech, 20597-1-AP), Apolipoprotein A1 (Abcam, ab52945), GM130 (Abcam, 52649), Calnexin (Proteintech, 10427-2-AP), GAPDH (KANGCHE, KC-5G5), Goat Anti-Mouse IgG HRP (KANGCHE, KC-MM-035), Goat Anti-Rabbit IgG HRP (KANGCHE, KC-RB-035), and IPKine™ HRP, Mouse Anti-Rabbit IgG LCS (Abbkine, A25022).

Nanoparticle tracking analysis (NTA)

Exosome samples were diluted in PBS for analysis. Exosome size distribution was measured by ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany) and analyzed by ZetaView 8.04.02 SP2 software.

Transmission electronic microscopy (TEM)

For TEM analysis, cells were fixed with 3% glutaraldehyde for at least 4 h. After being washed by PBS for three times, samples were post-fixed using 1% osmic acid for 2 h. Then, samples were washed by PBS for three times again and sequentially dehydrated in 50%, 70%, 80% and 90% alcohol for 15 min. Next, samples were dehydrated in 100% alcohol and 100% acetone twice for 15 min, respectively. After samples were embedded using epoxy resin to form small blocks, ultrathin sections (100 nm) of samples were harvested by an ultramicrotome (UC7, Leica, Germany). Ultrathin sections were stained with uranyl acetate for 20 min and were stained with lead citrate for 15 min. Finally, samples were imaged by TEM (Tecnai G2 Spirit, USA).

For TEM analysis of exosomes, the isolated exosomes (10 μl) were fixed in 4% paraformaldehyde (PFA, Beyotime, P0099-500 ml), then were dropped onto formvar-carbon-coated grids. After 5 min, the excess fluid in grids was removed. Next, samples were stained with 3% phosphotungstic acid for 5 min. Finally, samples were air-dried and were analyzed by TEM (Tecnai G2 Spirit, USA).

Confocal imaging

Cells on coverslips were fixed by 4% PFA at room temperature for 20 min and washed by PBS with 2 mg/ml glycine (GenStar, VA13110-500 g). Then, samples were treated with 0.3% Triton X-100 for 10 min and blocked by 10% FBS for 1 h at room temperature. After being washed by PBS, samples were incubated in primary antibody which was diluted in PBS containing 2% FBS for 1.5 h at room temperature. Next, samples were incubated in secondary antibody followed by 4’, 6-diamidine-2-phenylindole (DAPI, Cell Signaling, 8961S). Finally, coverslips were mounted onto slips using fluorescence mounting medium (Dako, S3023) and fluorescent images were taken by immunofluorescence microscope (Zeiss 710 NLO, Germany).

Antibodies used for immunofluorescence included Flag M2 (Sigma-Aldrich, F1804), PTRF (Proteintech, 18892-1-AP), Alexa Fluor 568 goat anti-mouse IgG (Invitrogen, A11004), Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, A11008).

RNA extraction and real-time PCR (RT-PCR) analysis

Total RNA was isolated from HeLa cells using TRIzol reagent (MRC, TR118-200). Complementary DNA (cDNA) was synthesized from the reverse transcription of 900 ng of total RNA using HiScript® II Q RT SuperMix for qPCR (Vazyme, R222-01) according to the product instruction. ChamQ SYBR qPCR Master Mix (Vazyme, Q311-02/03) was used for RT-PCR. Samples were detected by a CFX96 Touch™ RT-PCR Detection System (Bio-Rad, USA). All primers used for RT-PCR were listed in Additional file 1: Table S1.

Statistical analysis

Statistical analyses were performed as the means ± SEM by GraphPad Prism 5. P values for differences were calculated using Student’s t-test. P \(<\) 0.05 was considered to a statistically significant difference.

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