IL)-24 is a tumor suppressor/cytokine gene that undergoes post-translational modifications (PTMs). Glycosylation and ubiquitination are important for IL-24 protein stabilization and degradation respectively. Little is known about IL-24 protein phosphorylation and its role in IL-24-mediated anti-tumor activities. In this study we conducted molecular studies to determine whether IL-24 phosphorylation is important for IL-24-mediated anti-cancer activity.Interleukin (IL-24-wild-type (IL-24wt) or IL-24 with all five phosphorylation sites replaced (IL-24mt) was used in the present study. Inhibition of tumor cell proliferation, cell migration and invasion, and induction of G2/M cell cycle arrest was observed in DOX-induced IL-24wt-expressing cells but not in IL-24mt-expressing cells. Secretion of IL-24mt protein was greatly reduced compared to IL-24wt protein. Further, IL-24wt and IL-24mt proteins markedly differed in their subcellular organelle localization. IL-24wt but not IL-24mt inhibited the AKT/mTOR signaling pathway. SiRNA-mediated AKT knockdown and overexpression of myristolyated AKT protein confirmed that IL-24wt but not IL-24mt mediated its anti-cancer activity by inhibiting the AKT signaling pathway.Human H1299 lung tumor cell line that was stably transfected with a doxycycline (DOX)-inducible (Tet-on) plasmid vector carrying the cDNA of Our results demonstrate that IL-24 phosphorylation is required for inhibiting the AKT/mTOR signaling pathway and exerting its anti-cancer activities. IL)-24 is a novel tumor suppressor and a member of the IL-10 cytokine superfamily [Interleukin  88, 101, and 161, and Threonine (Thr) 111 and 133. Using molecular techniques, we replaced all of the five phosphorylation sites, producing a mutant (IL-24mt). We compared IL-24mt with wild-type IL-24 (IL-24wt). New to science, our data show that IL-24 phosphorylation is required for IL-24-mediated anti-cancer activities.In the present study, we investigated whether IL-24 phosphorylation is required for antitumor activities. The human The present study provides a platform for identifying the phosphorylation site(s) critical for IL-24 to function as an anti-cancer drug. Studies investigating the molecular mechanisms of IL-24 phosphorylation are also warranted.wt-expressing H1299 cells showed a typical expression pattern [mt-expressing cells showed a single 19–20 Kd protein band, suggesting that phosphorylation regulates IL-24 protein maturation.IL-24 pattern , 11 withwt and IL-24mt protein. We detected 26 Kd bands in sample lysates prepared from IL-24wt-expressing cells, reflecting IL-24wt protein , and secretory organelles [wt protein localized to the cytoplasm, ER, and Golgi. However, IL-24mt protein was primarily localized to the perinuclear area and overlapped with ER  promoter  compared with controls  compared with controls (8.05%). These results demonstrate that phosphorylation is required for IL-24wt-mediated G2/M-phase cell-cycle inhibition.Previous studies reported that IL-24poptosis , 5, 17. wt- and IL-24mt-expressing H1299 cells. We observed activated caspase-9, PARP, and phosphorylated (p) JNKThr183/Tyr185 only in IL-24wt-expressing cells, indicating that phosphorylation is required for the pro-apoptotic activity of IL-24 -IL-24 induces apoptosis in cancer cells by activating caspase-9, poly (ADP ribose) polymerase (PARP), and JNK , 17–19. 4 Figure .wt cells.We previously demonstrated that lung cancer cells expressing exogenous wild-type IL-24 migrate and invade less . Here, wwt-expressing cells migrated into 26.5% of the scratched area, whereas IL-24mt-expressing cells migrated into 94% of the scratched area  pathway (data not shown). Thus, we investigated the regulation of the AKT-mTOR pathway by IL-24. Induction of IL-24wt in H1299-wtIL24 cells significantly decreased pAKTS473 protein expression at 24 h and 48 h  analysis , 22 on Ih Figure . Expressn Figure . Furtherh Figure . The exph Figure . HoweverT308 expression and on AKT isoforms AKT1 and 2. AKT1 and 2 are ubiquitously expressed and individually contribute to cell survival, proliferation, and metastasis via different mechanisms [wt-expressing cells, we observed a marked reduction in pAKTT308, pAKT1S473, and pAKT2S474 expression at 48 h, compared with controls. IL-24mt expression increased pAKTT308 and pAKT2S474, with no change in pAKT1S473, suggesting that IL-24 phosphorylation is involved in the regulation of AKT isomers  at amino acid 246 (T246A), such that AKT cannot phosphorylate PRAS40, resulting in reduced pPRAS40 expression and mTOR activation. We anticipated that induction of IL-24wt, along with mutated PRAS40 expression, would show greater reduction in pPRAS40 expression. PPRAS40T246 expression was significantly reduced in mutant PRAS40T246A plasmid-transfected cells compared with controls  246 inactivates PRAS40 and influences mTOR activation [e Figure . Our reswt expression reduced AKT-mediated PRAS40 inactivation. IL-24wt expression might, then, increase PRAS40 inhibition of mTORC1 by increasing the tight interaction between PRAS40 and Raptor. To test this, we treated H1299-wtIL24 cells with DOX for 48 h. The cytosolic extracts were immunoprecipitated with PRAS40 and immunoblotted for Raptor. We observed a marked reduction in Raptor binding to PRAS40 upon IL-24wt induction  for 48 h -mediated substrate access to Raptor , 40. Sins Figure . To detes Figure . While wwt reduced the phosphorylation of GSK-3 and increased β-catenin phosphorylation  that are critical for IL-24 to function as an anti-cancer drug.We cultured human non-small cell lung cancer cells (H1299) in RPMI 1640 medium (GIBCO) as previously described [MDA-7/IL-24  cDNA is 621 bp in size with 206 amino acids. Potential phosphorylation sites of MDA-7/IL-24 protein predicted by NIH NCBI ORF finder are S88, S101, T111, T133 and S161 where S stands for Serine; T stands for Threonine. The software predicted that phosphorylation in MDA-7/IL-24 are as following: amino acid 88 SAR, amino acid 133 TLK, amino acid 161 SIR are casein kinase II consensus sites; amino acid 101 SDAE, amino acid 111 TLLE, amino acid 161 SIRD are protein kinase C (PKC) consensus sites [IL-wt24 cDNA cloned in pLJ143 plasmid backbone was used as a template to create the IL-24 phosphorylation mutant (mtIL-24). Briefly, IL-wt24 cDNA was retrieved by BamHI and XhoI restriction enzyme digestion and ligated back into to pBlueScript SK+ (Agilent). The sequence was verified by polymerase chain reaction (PCR) using primers MDA-7.F.BamHI (forward primer) and MDA7.R.XhoI  plasmid was generated by replacing the three serine (S) and two threonine (T) sites using the forward and reverse PCR primers , are used as templates for overlaying both segments together using previous 5′ forward primer and next 3′ reverse primer. The resulting PCR product with all five phosphorylation site replaced was subsequently TA cloned into TOPO plasmid vector (Invitrogen) and sequenced at the DNA sequencing core facility .On the basis of wild-type rs Table  and the rs Table  for creawtIL-24 and mtIL-24 cDNA from the TOPO plasmid vector was sub-cloned into pcDNA3.1+ plasmid expression vector (Invitrogen). The resultant plasmids were verified by BamHI-XhoI restriction enzyme digestion, PCR, and by the inserted signature restriction enzyme digestion. All PCR reactions performed in the study followed the High Fidelity PCR system protocol (Roche). Restriction enzymes used in the study were purchased from New England Biolabs. The pcDNA3.1+ plasmid vector carrying wtIL-24 and mtIL-24 cDNA sequence were either used in transient transfection studies or used in creating doxycycline (DOX)-inducible plasmid vector as described below.The wtIL-24 and mtIL-24 cDNA sequence cloned in pcDNA3.1+ plasmid vector was retrieved by BamHI and PmeI restriction enzyme digestion and sub-cloned into BamHI and EcoRV enzyme digested pTRE-Tight plasmid (Clontech). The resulting pTre-wtIL24 and pTre-mtIL24 plasmids with appropriate inserts were verified by PCR, restriction enzyme digestions and signature enzyme digestions. The plasmids were subsequently used for creating Tet-ON inducible stable cell lines per manufacturer's guidelines. Briefly, human H1299 lung cancer cell line was transfected with pTet-ON-adv plasmid (Clontech) using Fugene (Roche) and selected in Neomycin  for fourteen days. Transfection studies were performed using 10% tetracycline free fetal bovine serum . The surviving fraction of cells was replated and transfected with pTreT-wtIL-24 or pTreT-mtIL-24 plasmid followed by hygromycin  for two weeks. The surviving fraction of cells were then expanded and screened for IL-24wt and IL-24mt expression by Western blotting after DOX (1 μg/ml) treatment for 24 h. The IL-24wt and IL-24mt expressing cells were then seeded in ninety-six-well plates and subjected to single-cell cloning. Each clonal population of cells was subsequently screened for IL-24 protein expression by Western blotting. The clone that demonstrated the highest IL-24wt and IL-24mt protein expression was expanded and labeled as H1299-wtIL24 and H1299-mtIL24 respectively and used in all of the studies described herein.The Tet-ON inducible system was purchased from Clontech. The wtIL24 and H1299-mtIL24 cells (1 − 2 × 105 cells/well) seeded in six-well plates were treated with DOX (1 μg/ml). The number of viable cells at 72 h after DOX treatment was determined by the trypan-blue exclusion assay method as described previously [H1299-eviously , 17.wtIL24 and H1299-mtIL24 cells (2 × 104) were mixed with 0.7% low-melting agarose and seeded in six-well tissue culture plates previously coated with 0.7% agarose. The plates were incubated at 37°C for 24 h. Then, the cells were treated with DOX (1 μg/ml). Incubation continued for fourteen days at 37°C, with culture medium replenished twice a week. The number of surviving colonies was determined by staining with 0.005% crystal violet. Colonies were photographed and counted.H1299-wtIL24 and H1299-mtIL24 cells (1 × 105) seeded in six-well plates were treated with DOX (1 μg/ml). Untreated cells served as controls. Cells were collected at 48 h after treatment and subject to flow-cytometric analysis as previously described [H1299-escribed , 17.wtIL24 and H1299-mtIL24 cells (1 × 105/well) seeded in six-well plates were trypsinized and collected at 24 h and/or at 48 h after DOX (1 μg/ml). Cell lysates were prepared and protein samples subjected to western blotting [S473, pAKTT308, AKT, pAKT1S473, AKT1, pAKT2S474, AKT2, pPRAS40T246, PRAS40, pmTORS2448, mTOR, pGSK-3S21/9, GSK-3, pβ-catenin S33/37/T41, β-catenin, pFLNaS2152, FLNa, pPAK1T423, PAK1, pJNKThr183/Tyr185, JNK, cyclin D1, caspase-9, and PARP  were purchased and used. Beta-actin (Sigma) was used as an internal loading control. Proteins were detected using the appropriate secondary antibodies, as previously described [H1299-blotting , 11, 17.escribed . Protein6) actively growing in 150-mm tissue culture plates were treated with DOX (1 μg/ml). Forty-eight hours after treatment, the cells were washed and trypsinized, and total cell lysates prepared. About 500 μg of total protein was mixed with 100 μl of appropriate antibody  and incubated at 4°C overnight with constant mixing. Immune complexes were captured by incubation with 75–100 μl protein A/G-coated agarose beads for 1 h at 4°C. The immunoprecipitated proteins were recovered by boiling the agarose beads in 2X SDS sample buffer and western blotting. The membrane was subsequently probed using the appropriate primary antibody , and the protein bands detected as previously described [Cells 5 × 10 activelyescribed .wtIL24 and H1299-mtIL24 cells (1 × 103) seeded in two-well glass chamber slides were treated with DOX (1 μg/ml). Twenty-four hours after treatment, the cells were washed with 1.0 mL warm 1X phosphate-buffered saline (PBS) at 37°C and subsequently fixed in 4% paraformaldehyde in PBS for 10 min at room temperature (RT). After fixation, the cells were washed with PBS, incubated with 100% methanol for 10 min, and permeabilized with 0.5% TritonX-100 for 10 min. The cells were washed with PBS, followed by incubation in blocking buffer  in PBS for 1 h at RT. Then, we added a mixture of anti-human IL-24 antibody raised in goat  and mouse anti-PDI antibody  in blocking buffer and incubated for 2 h at RT. The cells were subsequently washed with 0.5 ml of 1X blocking buffer, followed by the addition of a mixture of Alexa Fluor® 488 (anti-mouse green) and Alexa Fluor® 594 (anti-goat red) diluted in 1 ml of blocking buffer. The cells were incubated in the dark for 1 h at RT, then counter stained with DAPI (Sigma) for 1 min. The stained cells were washed with 1X PBS, mounted with Dako cytomation fluorescent mounting media (Dako), and then viewed using a laser confocal microscope equipped with appropriate fluorescein filters.H1299-wtIL24 and H1299-mtIL24 cells (1 × 104) seeded in six-well plates were incubated overnight at 37°C, 5% CO2. The next day, the culture medium was replaced with fresh RPMI-1640 medium containing 2% FBS. DOX (1 μg/ml) was added. Culture supernatants were collected at different times after treatment, centrifuged, and stored at −80°C. We detected IL-24 protein in the supernatant by sandwich ELISA (R&D Systems). Briefly, ninety-six well flat-bottomed plates were coated with 100 μl of anti-IL-24 capture antibody  overnight at +4°C. The plates were washed with sterile PBS followed by incubation in 100 μl of blocking buffer containing 10% bovine serum albumin (BSA in PBS) for 1 h at RT. On completion of incubation, the blocking buffer was aspirated and 100 μl of culture supernatant collected from H1299-wtIL24 and H1299-mtIL24 cells with and without DOX treatment was diluted in PBS (1:2) and added in duplicate wells and incubated for 2 h at RT. The culture medium from the wells was subsequently aspirated and washed with sterile PBS. To the wells 100 μl of horse-radish peroxide (HRP)-conjugated anti-IL-24 detection antibody (R&D Systems) was added. After 1 h incubation, the wells were washed with sterile PBS followed by addition of streptavidin-horse radish peroxidase (100 μl/well) and incubation in the dark for 20 min at RT. The reaction was stopped by adding 50 μl of stop solution (R&D Systems). The plates were then read using a plate reader equipped with 450 nm excitation and 540 nm emission filter.H1299-in vitro scratch assay was performed using the CytoselectTM 24-well wound healing assay (Cell Biolabs). Briefly, H1299-wtIL24 and H1299-mtIL24 cells (2.5 × 104) were added to 24-well plates with inserts in place. After 24 h incubation, we removed the inserts to generate a 0.9 mm wound field, washed it with media, and incubated at 37°C. The cells were then treated with DOX (1 μg/ml). We photographed the scratch area 24 h later using a Nikon TiU microscope (Nikon). The distances between the two cell edges were measured per the manufacturer's instructions.The wtIL24 and H1299-mtIL24 cells (5 × 104) suspended in 1 ml RPMI-1460 medium were seeded in the upper chambers  and placed in a six-well plate filled with serum-free RPMI-1640 medium (lower chamber). After 24 h, the culture medium was replaced with fresh medium containing 20% tetracycline-free FBS . The upper chamber was filled with 2% tetracycline-free FBS-containing medium, with or without DOX . Following incubation for 24 h and 48 h, the inserts were removed and processed as previously described [H1299-escribed , 52. FouwtIL24 and H1299-mtIL24 (5 × 104) cells were seeded into the upper chambers. The lower chambers were filled with serum-free medium. After 24 h, the medium was replaced with 20% FBS-containing culture medium, and the upper chambers were filled with 2% FBS-containing culture medium, with or without DOX (1 μg/ml). After an additional 24 h and 48 h incubation, the filters were removed and processed as previously described [The cell invasion assay was performed using six-well Matrigel invasion chambers with 8-μm pore inserts (BD Biosciences). H1299-escribed , 52. TumIL24 (wt and mt) cells (1 × 105 cells/well) were seeded in serum-free medium in six-well plates and transfected with 100 nM AKT siRNA (Santa Cruz) using transfection reagent (Dharmacon), per the manufacturer's instructions. Six hours later, the medium was replaced with RPMI-1460 containing 2% tetracycline-free serum, with or without 1 μg/ml DOX. Untransfected cells served as a control. After 48 h of incubation, the cells were harvested and total cell lysates prepared for western blot analysis.H1299-IL24 (wt and mt) cells (5 × 104) were seeded in the upper chamber of the inserts and transfected with AKT siRNA (100 nM), with or without 1 μg/ml DOX. The lower chamber was filled with 20% tetracycline-free FBS-containing medium. We counted the number of migrated cells 48 h after treatment.For the cell migration assay, H1299-wtIL24 cells (1 × 105 cells/well) were seeded in six-well plates and transfected with 0.5 μg myr-AKT or pPRAS40T246 DNA (Addgene) using Lipofectamine 2000 (Invitrogen), per the manufacturer's instructions. Six hours after transfection in serum-free medium, the medium was replaced with RPMI-1460 containing 2% tetracycline-free serum, with or without 1 μg/ml DOX. Untransfected cells served as controls. After 48 h of incubation, the cells were harvested and total cells lysate subjected to western blotting.H1299-wtIL24 cells (5 × 104) were seeded in the upper chamber of the inserts and were transfected with myr-AKT plasmid (0.5 μg), with or without 1 μg/ml DOX. The rest of the procedure followed the description under “cell migration assay”.For the cell migration assay, H1299-SD. Data were analyzed using two-tailed student's t test or one-way analysis of variance (ANOVA) using SPSS 12.0 . P < 0.05 was considered statistically significant.All experiments were performed at least twice. Unless otherwise stated, all data are shown as mean ±


The resistance of apoptosis in cancer cells is pivotal for their survival and is typically ruled by mutations or dysregulation of core apoptotic cascade. Mantle cell lymphoma (MCL) is a non-Hodgkin's B-cell malignancy expressing higher anti-apoptotic proteins providing survival advantage. B-PAC-1, a procaspase activating compound, induces apoptosis by sequestering Zn bound to procaspase-3, but the amino acids holding Zn in Caspase-3 is not known. Here we show that reintroduction of WT caspase-3 or 7 in Caspase3–7 double knock-out (DKO) mouse embryonic fibroblasts (MEF) promoted B-PAC-1 to induce apoptosis (27–43%), but not in DKO MEFs or MEFs expressing respective Casp3–7 catalytic mutants (12–13%). Using caspase-6 and -9 exosite analysis, we identified and mutated predicted Zn-ligands in caspase-3  and overexpressed into DKO MEFs. Mutants carrying E272A abrogated Zn-reversal of apoptosis induced by B-PAC-1 via higher XIAP and smac expressions but not in H108A or C148S mutants. Co-immunoprecipitation analysis revealed stronger XIAP-caspase-3 interaction suggesting a novel mechanism of impulsive apoptosis resistance by disrupting predicted Zn-ligands in caspase-3. B-PAC-1 sponsored apoptosis in MCL cell lines (30–73%) via caspase-3 and PARP cleavages accompanied by loss of Mcl-1 and IAPs including XIAP while Zn substantially abrogated B-PAC-1-driven apoptosis (18–36%). In contrary, Zn is dispensable to inhibit staurosporin, bendamustine, ABT199 or MK206-induced apoptosis. Consistent to cell lines, B-PAC-1 stimulated cell death in primary B-lymphoma cells via caspase-3 cleavage with decline in both Mcl-1 and XIAP. This study underscores the first genetic evidence that B-PAC-1 driven apoptosis is mediated via Zn chelation. Mantle cell lymphoma (MCL) is a fatal B-cell non-Hodgkin's malignancy with a tendency to disseminate throughout the body, including lymphoid tissues, bone marrow and peripheral blood there by offering a broad spectrum of clinical, pathological, and biological significance. Cyclin D1 over-expression owing to reciprocal translocation event t11;14)q13;q32) [ 2–5]. A. A5]. Al;14q13;q3Procaspase-activating compound-1 (PAC-1) was discovered as the first line of procasp3–7 activators along with other PAC-1 derivatives , 14–16. 50 value, we selected 10 μM for all additional experiments unless specied otherwise. Co-incubation of B-PAC-1 with Zn rescued cell death in these cells entailing that Zn plays a role in B-PAC-1-induced PCD . Interestingly, both Annexin V-PI FACS analysis and protein analysis revealed that ATM deficient  Granta-5Further explication of the role of Casp3 and Casp7 during B-PAC-1-induced apoptosis was evaluated in a series of MEF cell lines. While B-PAC-1 (10 μM for 24 hr) sensitized WT MEF (Casp3-7+/+) to PCD, Zn (100 nM for 24 hr), mostly inhibited B-PAC-1 action. In contrast, MEF cells lacking a single allele for both Casp3 and -7 (Casp3-7+/−) were less sensitive to B-PAC-1-driven apoptosis compared with WT MEF. As expected B-PAC-1 failed to sensitize DKO MEF cells to induce PCD, suggesting dependence of Casp3 and -7 for final execution of PCD by B-PAC-1 Figure .Stable reintroduction of WT Casp3 into DKO MEF cells significantly sensitized B-PAC-1 induced PCD, while Casp3 catalytic mutant (Casp3C163A) blunted the effect of B-PAC-1 suggesting that Casp3 with an active site cysteine in the nucleophilic attack site is required for targeting B-PAC-1 to induce PCD. Although less sensitive, B-PAC-1 still induced PCD in DKO MEF cells stably transfected with WT Casp7 but not in Casp7 catalytic mutant (Casp7C186A) reconfirming that both Casp3 and -7 are important watchdogs for the final execution of B-PAC-1-induced apoptosis Figure . WesternActive Casp3 is a dimer of two cleaved procaspases as illustrated in Figure 50 values were titrated using WT Casp3 transfected DKO MEF cells  in both WT and H108A mutant, while C148S and mutants carrying E272A were resistant to 066. This data prompted us to study real time RT-PCR analysis of Casp3 and XIAP mRNA levels in these clones. A relatively higher Casp3 mRNA expression was observed in H108A, C148S and in C148S+E272A double mutant compared with WT , MZL (n = 5), DLBCL (n = 4) and FL (n = 3) were tested , however, the Zn chelation is not widespread with other apoptosis-inducing drugs  through a cascade of events  leading Recently we documented B-PAC1 efficacy to induce apoptosis in both CLL and multiple myeloma cells , 43, howWhile the active sites for Zn in Casp3 molecule is unknown, we used the exosite cluster analysis of Casp9  and muta2 of S36 in the large subunit. The carboxyl group of E272 is located within hydrogen bonding distance to H234 in the opposing small subunit in the dimeric unit. This network of interactions likely plays a role in stabilization of helix 231–247 and may be important for interaction with N-terminal segment of the large subunit. Replacement of E272 with alanine would remove the hydrogen bond with Y274 which could destabilize this cluster and alter the interactions between the large and small subunits of heterodimer. Further, disruption of this cluster could interrupt the interactions between the two small subunits thereby destabilizing the tetrameric structure of the enzyme complex which could alter the placement of the loop bundle [E272 is involved in several intra- and inter-domain interactions that may account for the observed effect Figure . Crystalp bundle  leading IAPs are unique molecules with BIR (baculovirus IAP repeat) domain and XIAP is the prototype member of the family. The complex structure of the Bir2 domain of XIAP and the catalytic domains of Casp3 indicates that the interaction takes place at the catalytic site and impact the loop bundle . Given tMycoplasma using a MycoTect kit (Invitrogen). MCL cell lines were validated by AmpF/STR Identification kit (Applied Biosystems) in the MD Anderson cell line validation core facility. Routine cell number and the mean cell volume were determined by Coulter channelyzer (Coulter Electronics).Three MCL cell lines  were used in this study. Granta-519 was maintained in Dulbecco modified Eagle medium with high glucose  supplemented with 20% FBS while both Jeko-1 and Mino cell lines were cultured in RPMI 1640 medium (ATCC) supplemented with 20% FBS. Wild type, heterozygote and double knockout mouse embryonic fibroblasts for Casp3 and Casp7 (DKO MEF) were maintained through passage 10 in DMEM (Cellgro) supplemented with 10% FBS. All media were supplemented with 1% penicillin-streptomycin (Invitrogen Inc.). These cell lines were routinely tested for For primary lymphomas, informed consent from every patient was obtained before inclusion in the study and was in accordance to the declaration of Helsinki. All protocols were approved by the Institutional Review Board at MD Anderson Cancer Center. Malignant lymphocytes from peripheral blood or from apheresis samples were isolated by Ficoll-Hypaque as described . Cells wB-PAC-1 and PAC-1a, were > 98% pure and were provided by Prof. PJ Hergenrother, University of Illinois , 16. SmaGCTAGCAAAAGGAGCAG-3′ for generating Casp3 H108-A mutation; 5′-TTTTTCAGAGGGGATCGTAGTAGAAG-3′ for generating Casp3 C148S mutation while Casp3 E272A mutation was introduced into a common reverse complement that contained a BglII cloning site (5′-CGATATCAGATCTATTTAGTGATAAAAAT AGAGTGCTTTTGTGA-3′). Mutated nucleotides are underlined. Similar two step PCR strategy was employed to create double (H108A and C148S or C148S and E272A) or triple  mutants. All plasmids were sequence verified at MD Anderson DNA sequencing core facility and cloned into EcoR1-BglII sites of p3XCMV-14 (Sigma).Plasmids 11813 (pcDNA3-Casp3-myc); 11814 (pcDNA3-Casp3 C163A-myc); 11815 (pcDNA3-Casp7-Flag); 11816 (pcDNA3-Casp7 C186A-Flag) and HA-XIAP (plasmid 25674: pEBB-HA-hILP) were obtained from Addgene. For Casp3–Zn mutation plasmids, a WT Casp3 construct was generated by cloning a PCR-amplified full-length human Casp3 coding region into EcoR1-BglII sites of p3XCMV-14 (Sigma) vector upstream of C terminal Flag epitope. Mutations in predicted Casp3-Zn binding amino acids were made by two-step PCR-based strategy. The following primers were used: 5′-GAAGATDKO MEF cells were transfected overnight with 3 μg of p3X-CMV FLAG-Casp3 WT or Casp3-Zn mutant plasmids or with an empty vector control by using Lipofectamine 2000 (Invitrogen Inc). Forty eight hour after transfection, cells were selected in G418 (500 μg/ml) containing medium for 3 weeks. Several independent colonies were isolated, expanded, and analyzed for protein expression. For transient co-transfection assay, HEK293 cells were transfected with 3 μg each HA-XIAP and p3X-CMV-FLAG Casp3 WT, Casp3-Zn-mutants or empty vector (p3X-CMV-FLAG plus pcDNA3.1) by using FuGENE6 (Promega Inc) transfection reagent. Forty eight hour after transfection cells were harvested for immunoprecipitation analysis.Sub-confluent stably over-expressed DKO MEF cells (in 100-mm plates) were lysed in IP buffer (Pierce) and an equal amount of lysates (3000 μg) was immunoprecipitated overnight with EZview red anti-Flag (25 μl/mg protein) affinity gel. For co-IP assay, HEK-293 cells were transfected with 3 μg each of HA-XIAP and p3XCMV-FLAG-Casp3 WT or Casp3-Zn mutant plasmids. At 48 hr post-transfection, cells were lysed in IP buffer and an equal amount of lysates (500 μg) was immunoprecipitated overnight with EZview red anti-Flag or EZview red anti-HA affinity gel (Sigma). Following immunoprecipitation, the beads were washed 3 times with lysis buffer, resuspended in 2X Laemmli sample buffer (Bio-Rad Lab), fractionated on SDS-12% polyacrylamide gel and analyzed by Western blot with rabbit anti-HA  or with mouse M2-FLAG (Sigma) antibodies.Protein lysates and immunoblots were prepared as described . ProteinTotal RNA from MEF cells was prepared  by using6 cells were stained with Annexin V FITC followed by propidium iodide staining and acquired on a FACS Calibur (BD Biosciences) and analyzed using Flow Jo software (TreeStar) as previously described [Briefly 10escribed .2O using standard protocol. Cells were mounted with ProLong Gold Antifade (Life Technologies Inc) and scanned in a Labophot-2 fluorescence microscope .Jeko-1 cells were cyto-spinned on slides  followint-test (paired) or by Anova analysis.Statistical analysis was done using Prism . All numerical results are presented as means ± SEM. The statistical significance of differences was analyzed using Student's


Re-evaluation of the phenotype of Vpu phosphorylation mutants and naturally occurring allelic variants reveals that the requirement for the Vpu phosphoserine motif in tetherin antagonism is dissociable from SCFβTRCP1/2 and ESCRT-dependent tetherin degradation. Vpu phospho-mutants phenocopy ExxxLV mutants, and can be rescued by direct clathrin interaction in the absence of SCFβTRCP1/2 recruitment. Moreover, we demonstrate physical interaction between Vpu and AP-1 or AP-2 in cells. This requires Vpu/tetherin transmembrane domain interactions as well as the ExxxLV motif. Importantly, it also requires the Vpu phosphoserine motif and adjacent acidic residues. Taken together these data explain the discordance between the role of SCFβTRCP1/2 and Vpu phosphorylation in tetherin antagonism, and indicate that phosphorylation of Vpu in Vpu/tetherin complexes regulates promiscuous recruitment of adaptors, implicating clathrin-dependent sorting as an essential first step in tetherin antagonism.HIV-1 Vpu prevents incorporation of tetherin (BST2/ CD317) into budding virions and targets it for ESCRT-dependent endosomal degradation via a clathrin-dependent process. This requires a variant acidic dileucine-sorting motif (ExxxLV) in Vpu. Structural studies demonstrate that recombinant Vpu/tetherin fusions can form a ternary complex with the clathrin adaptor AP-1. However, open questions still exist about Vpu’s mechanism of action. Particularly, whether endosomal degradation and the recruitment of the E3 ubiquitin ligase SCF βTRCP1/2 that binds to phosphorylated serine residues in the Vpu cytoplasmic tail. Again, discrepancies exist about the importance of this interaction in tetherin’s counteraction. Here we show that Vpu phosphorylation, in combination with its physical interaction with tetherin, regulates interaction with both AP-1 and the other major cellular clathrin adaptor, AP-2. These interactions can be decoupled from SCFβTRCP1/2 recruitment, thus indicating clathrin-dependent mis-trafficking as a critical step in tetherin antagonism by Vpu. Additionally, the ability to interact both with AP-1 and AP-2 in a tetherin-dependent manner indicates a redundancy in host cofactors used by Vpu that explains disparate previous observations of its mechanism of action.Counteraction of tetherin, a host antiviral protein that blocks viral release from infected cells, is an essential attribute of HIV-1 and its related viruses. The HIV-1 accessory protein Vpu binds to tetherin, preventing its incorporation into viral particles, and targets it for ubiquitin-dependent degradation. This involves mis-trafficking of tetherin by a Vpu-dependent mechanism through the engagement of clathrin adaptor proteins. Although structural evidence exists for Vpu and tetherin interacting with clathrin adaptor 1 (AP-1), evidence that it is required for Vpu-mediated tetherin counteraction is still lacking. Tetherin degradation by Vpu also requires an E3 ubiquitin ligase, SCF Much of this discrepancy may be attributable to whether assays are performed in virally infected cells or those transiently transfected with Vpu, tetherin or both [Tetherin recycles to the PM via the trans-Golgi network (TGN) . This re or both . While E or both , evidenc or both . Further or both ,25,31. M or both . Indeed  or both . This is or both .vice versa) and AP-2 (clathrin-dependent endocytosis from the PM) . In keeiewed in ,38. Mutaiewed in . In partiewed in . A receniewed in . In fusiiewed in . Some deiewed in . Neitheriewed in . Added tiewed in ,40. Likeiewed in ,35, and iewed in ,42.AP1 binding to a non-canonical acidic dileucine motif in CI-M6PR has been associated with upstream serine phosphorylation by CKII previously . Thus weβTRCP1/2 E3 ligase and the ultimate degradation of tetherin to the counteraction of its physical antiviral activity by Vpu has been controversial. Since the discrepant studies were mostly performed under conditions of transient transfection of tetherin, provirus or both, and which have been shown previously to lead to artifactual effects on tetherin degradation [The importance of the SCFradation , we re-eradation ,36. Despradation  and blocradation , and allA previous study indicated that HRS interacted with Vpu in immuno-precipitates . We confβTRCP1/2 complex, under conditions when it is sufficiently depleted to block this, there is no effect on Vpu-mediated tetherin antagonism.We next similarly re-evaluated the effect of simultaneously knocking down β-TrCP1 and 2 on Vpu-mediated tetherin-degradation and tetherin-counteraction in infected cells. Again despite efficient knockdown, we saw little effect of this treatment on HIV-1 WT release . Of noteμ. We found similar Vpu mutants with tetherin-defective phenotypes in our patient cohort [41I42/L45I46 in the first alpha helix to alanines in the NL4.3 provirus led to a profound defect in tetherin antagonism and degradation without preventing interaction  and serine/threonine (the proposed SCFion site ) mutatioFinally we examined mutations within the DSGNES motif itself. The consensus for a β-TrCP-binding site is DSGxxS, yet the N55/E56 in group M Vpu is almost universally conserved. We found rare mutations (N55H/E56G) in patients that displayed impaired tetherin antagonism despite retaining β-TrCP interaction . Similarin vivo by conventional immunoprecipitations. To examine Vpu interaction with AP-1, we initially employed a proximity-based biotin ligase assay  from subversion of trafficking (counteraction) suggests that the importance of the former might reflect downstream consequences of tetherin restriction. Enhanced antagonism of the long tetherin isoform by Vpu could be required because of its signal transduction or its ability to deliver retained virions to endosomes ,40. Our HEK293T cells were obtained from ATCC (American Tissue Culture Collection). 293T tetherin cell lines stably expressing human tetherin and mutants were previously described ,57. The Primary human CD4+ T cells were isolated from fresh venous blood drawn from healthy volunteers. CD4+ T cells were purified from total peripheral blood mononuclear cells (PBMC) isolated by lymphoprep (AXIS-SHIELD) gradient centrifugation using a CD4+ T cell Dynabeads isolation kit (Invitrogen). T cells were then activated for 48 h using anti-CD3/anti-CD28 magnetic beads (Invitrogen). The beads were then removed cells were then maintained in rhIL-2 (20 U/ml) (Roche).For full-length HIV-1 WT, HIV-1 ΔVpu, HIV-1 Vpu LILI, HIV-1 Vpu ELV, HIV-1 Vpu LILI/ELV, HIV-1 Vpu 2/6A, HIV-1 Vpu 2/6A/ELV virus stocks pseudotyped with the Vesicular Stomatitis Virus Glycoprotein (VSV-G), 293T cells were transfected with 2 μg of proviral plasmid in combination with 200 ng of pCMV VSV-G. 48 hours post-transfection, the supernatant containing virions was harvested and endpoint titers were determined on HeLa-TZMbl cells as described previously [2 (Sigma) were added to the composition of 8% polyacrylamide gels to induce mobility shifts in phosphorylated proteins, to final concentrations of 25 μM and 50 μM, respectively.For virus release assays using transient transfection, subconfluent 293T cells or derivatives were plated in 24 well plates and transfected with 500 ng of NL4.3 proviral plasmid, in combination with increasing concentrations of tetherin  and fixed 25 ng of Vpu-HA or mutants using 1 μg/ml polyethyleneimine (Polysciences). Medium was replaced 8 hours post-transfection and cells and supernatants were harvested after 48 hours. The infectivity of viral supernatants was determined by infecting HeLa-TZMbl and assayed for β-galactosidase activity as previously described . For bio5 293T tetherin cells were infected with VSV-G-pseudotyped HIV-1 WT, HIV-1 ΔVpu, HIV-1 Vpu LILI, HIV-1 Vpu ELV or HIV-1 Vpu 2/6A at an MOI of 2. The medium was replaced 4 hours after infection. 48 hours post infection cell lysates were harvested and subjected to SDS-PAGE and Western blotted for rabbit anti-HSP90 (Santa Cruz Biotechnologies) and polyclonal rabbit anti-tetherin antibody (kindly provided by K Strebel through the NIH ARRP) [1.5 x 105 cells per well in a 12 well plate. After 6 hours, the first transfection was performed. For each well, 2 μl Dharmafect (Thermo Scientific) was added to 98 μl of Opti-MEM (Life Technologies), this solution was added to 5 μl of 20 μM siRNA in 95 μl of Opti-MEM according to manufactures protocol. For HRS knockdown, siRNA oligonucleotide against HGS targeting the CCGGAACGAGCCCAAGTACAA sequence (Qiagen) was used. For UBAP1 knockdown, siRNA oligonucleotide against UBAP1 targeting CTCGACTATCTCTTTGCACAT (Qiagen) was used. For TSG101 knockdown, siRNA oligonucleotide sequence CCUCCAGUCUUCUCUCGUCUU (Thermo Scientific) was used. For β-TrCP1 and 2 knockdown, SMARTpool siRNA against human BTRC and FBXW11 (Thermo Scientific) were used. A non-targeting siRNA was used as control (Thermo Scientific). The cells were re-seeded into a 24 well plate on day 2 and a second transfection was performed according to manufactures protocol. The cells were infected 3 hours post transfection with VSV-G-pseudotyped HIV-1 WT, HIV-1 ΔVpu at an MOI of 0.8. The infectivity of viral supernatants was determined by infecting HeLa-TZMbl as described above. Cell lysates and viral particles were subjected to SDS-PAGE, and Western blot assays were performed using a rabbit polyclonal anti-HRS (HGS) antibody (Millipore), a polyclonal rabbit anti-UBAP1 antibody (Proteintech) and a monoclonal mouse anti-TSG101 antibody (Abcam).293T tetherin cells were seeded at a density of 2 x 10HeLa-TZMbl cells were transfected with 400 ng of pCR3.1 GFP and 400 ng of pCR3.1 Vpu-HA or indicated mutants. 48 hours post transfection the cells were harvested and stained for surface tetherin using a monoclonal anti-BST2 IgG2a antibody (Abnova) and a goat-anti-mouse IgG2a-Alexa633 conjugated secondary antibody . Tetherin expression on GFP positive cells was then analyzed using a BD FACSCanto II flow-cytometer (Becton Dickinson) and the FlowJo software.For Vpu/HRS coIP, 293T tetherin cells were transfected with 700 ng of pCR3.1 myc-HRS or indicated mutants/truncations in combination with pCR3.1 Vpu-HA or indicated mutant or pCR3.1 GFP expression plasmids. 48 hours post transfection the cells were lysed in buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 200 μM sodium ortho-vanadate, 5 mM NEM, complete protease inhibitors (Roche) and 1% digitonin. After removal of the nuclei, the supernatants were immunoprecipitated with 5 μg/ml monoclonal mouse anti-myc antibody previously described . Western blot assays were performed using a polyclonal rabbit anti-HA antibody (Rockland) and rabbit polyclonal anti-HRS (HGS) antibody (Millipore). For Vpu/tetherin coIP, 293T cells were transfected twice over 48 hours with siRNA oligonucleotide against UBAP1 targeting CTCGACTATCTCTTTGCACAT or Non-targeting siRNA was used as control (Dharmacon). The cells were then infected with VSV-G-pseudotyped HIV-1 WT, HIV-1 ΔVpu, HIV-1 Vpu LILI or HIV-1 Vpu A14L W22A at an MOI of 2. 48 hours post infection the cells were lysed on ice for 30 min in buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, complete protease inhibitors (Roche) and 1% digitonin . Immunoprecipitation was performed as previously described  and WestHela cells were grown on coverslips, transfected with 50 ng of pCR3.1 Vpu-HA or indicated mutant. 16 hours later cells were fixed in 4% paraformaldehyde/ PBS, washed with 10 mM glycine/ PBS, and permeabilized in 1% bovine serum albumin/ 0.1% Triton-X100/ PBS for 15 min. Cells were stained using anti-rabbit polyclonal HA antibody (Rockland) in combination with sheep anti-human TGN46 (AbD Serotec), followed by the appropriate secondary antibodies conjugated to Alexa 488 or 594 fluorophores . Cells were mounted on glass slides using ProLong AntiFade- 4’,6-diamidino-2-phenylindole (DAPI) mounting solution  and images were captured with a Nikon ESCLIPSE Ti inverted microscope. Z stacks were taken of all cells, images deconvolved using AutoQuant X3 and analyzed using the ImageJ software.293T, 293T tetherin, 293T tetherin Y6,8A or 293 Rhesus tetherin cells were transfected with 8 μg GFP expression construct, pCR3.1 Vpu-HA or mutant thereof. Transfection media was changed 6 hours post transfection and cells incubated with 50 nM concanamycin. In the case of CKII inhibitor treatment, cells were treated with 50 μM final Tyrphostin 24 hours prior to harvesting. 48 hours post transfection, cells were trypsinised and washed in PBS. Cells were cross-linked with 0.05% HCHO/PBS for 10 min at 37°C. The cross-linking reaction was then quenched by incubating cells in 0.25 M glycine for 5 min. Cells were washed once in PBS before resuspension in lysis buffer ). Cells were lysed on ice for 10 min followed by repeated sonication (3 x 10 s cycles with 20 s rests). The cell lysates were clarified by centrifugation at 1000 x g for 10 min and supernatants were immunoprecipitated with 5 μg/ml mouse monoclonal anti-HA.11 antibody (Covance) or rabbit polyclonal anti-HA antibody (Rockland) on Dynabeads protein G beads (Life Technologies) for 4 hours at 4°C. Beads were collected post incubation and washed 5 times in lysis buffer before cross-links were reversed in 1% SDS, 10 mM EDTA and 5 mm DTT at 65°C for 45 min. Western blot assays were performed using rabbit polyclonal anti-HA antibody (Rockland), polyclonal rabbit anti-tetherin antibody, mouse monoclonal anti-HA.11 antibody, mouse monoclonal anti-AP-1γ1 antibody (Sigma) and mouse monoclonal anti-AP-2α antibody (Sigma). Vpu/β-TrCP2 cross-linking IP was previously described by . 293T or Permission to isolate primary human CD4+ T cells from healthy consenting donors was provided by the KCL Infectious Disease BioBank Local Research Ethics Committee, reference SN-1/6/7/9.S1 Fig(A) Schematic representation of HRS C-terminal truncations. (B) 293T cells were transfected with pCR3.1 Vpu-HA in combination with a pCR3.1 myc-HRS or myc-HRS truncation expression vector. 48 hours post transfection cells were lysed and immunoprecipitated with anti-myc antibody. Total cell lysates and precipitates were subjected to SDS-PAGE and analysed by Western blotting for myc-HRS and Vpu, and analyzed by ImageQuant. Asterisk: anti-HA antibody heavy chain (C) Immunoprecipitation was performed as in (B) but with myc-HRS W25A L29D and myc-HRS A266Q A268Q mutants.(TIF)Click here for additional data file.S2 Figwww.hiv.lanl.gov). (B) 293T cells were transfected with NL4.3 HIV-1 WT, ΔVpu, Vpu LILI, Vpu ELV or Vpu 2/6A mutant together with increasing concentrations of pCR3.1 tetherin-HA expression plasmid. Cell lysates and sucrose purified viral supernatants from 50 ng tetherin input were subjected to SDS-PAGE and analyzed by Western blotting for HSP90, HIV-1 p24CA and Vpu, and analyzed by LiCor quantitative imager. Asterisk: non-specific band. (C) Infectivity of viral supernatants was assayed on HeLa-TZMbl reporter cells. Infectious virus release was plotted as β-galactosidase activity in relative light units (RLU). Error bars represent the standard deviation of three independent experiments. (D) HeLa-TZMbl cells were co-transfected with pCR3.1 Vpu-HA or indicated mutant and a GFP expression vector. Cell-surface tetherin levels were analysed 48 hours post transfection by flow cytometry. GFP positive cells were gated and tetherin levels (solid lines) were compared to un-transfected cells or transfected with indicated Vpu (dotted lines). Numbers indicate median fluorescence intensities of endogenous tetherin surface levels. The solid peak in the upper histogram in the middle of the panel represents binding of the isotype control. (E) 293T tetherin expressing cells were transfected twice over a 48 hour period with siRNA oligonucleotides directed against UBAP1 or non-targeting control. The cells were then infected with HIV-1 WT, HIV-1 Vpu LILI, HIV-1 Vpu A14L W22A or HIV-1 ΔVpu at an MOI of 2. 48 hours later, cells were lysed and immunoprecipitated with anti-tetherin antibody. Total cell lysates and precipitates were subjected to SDS-PAGE and analyzed by Western blotting for tetherin, UBAP1 and Vpu, and analyzed by ImageQuant.(A) LogoPlots of the first alpha helix of the Vpu cytoplasmic tail from HIV-1 subgroup M clades A, B, C, D, G and H generated from sequences obtained from the Los Alamos database ((TIF)Click here for additional data file.S3 Fig(A-B) 293T, 293T tetherin or Y6,8A tetherin cells were infected with VSV-G pseudotyped NL4.3 wt or mutant virus at an MOI of 0.8. (A) 48 hours post infection viral supernatants were assayed for infectivity using HeLa-TZMbl reporter cells as in (TIF)Click here for additional data file.S4 Fig(A) 293T tetherin cells were transfected with NL4.3 ΔVpu proviral plasmid in combination with YFP expression vector and pCR3.1 2_87 Vpu or mutants thereof. 48 hours post transfection infectivity of viral supernatants was determined on HeLa-TZMbl cells as in (TIF)Click here for additional data file.S5 Fig(A to D) HeLa-TZMbl cells were co-transfected with pCR3.1 Vpu-HA or indicated mutant and a GFP expression vector. Cell-surface tetherin levels were analyzed 48 hours post transfection by flow cytometry. GFP positive cells were gated and tetherin levels (solid lines) were compared to un-transfected cells or transfected with indicated Vpu (dotted lines). Numbers indicate median fluorescence intensities of endogenous tetherin surface levels. The solid peak in the upper histogram in the middle of the panel represents binding of the isotype control.(TIF)Click here for additional data file.S6 Fig(A) Schematic representation of proximity-based biotin ligase assay. (B) 293T tetherin cells were transfected with NL4.3 ΔVpu proviral plasmid in combination with YFP expression vector and pCR3.1 Vpu or indicated mutant thereof. 48 hours post transfection infectivity of viral supernatants was determined on HeLa-TZMbl cells as in (TIF)Click here for additional data file.S7 Fig(A) 293T cells were transfected with pCR3.1 Vpu-HA, Vpu ELV-HA, Vpu 3/7A-HA, Vpu LILI-HA or Vpu NE-HA mutants. (B) 293T tetherin Y6,8A were transfected with pCR3.1 Vpu-HA, Vpu A15L/W23A-HA, Vpu ELV-HA, Vpu 3/7A-HA, Vpu LILI-HA or Vpu NE-HA mutants. 48 h post transfection, cells were lysed and cross-linked using PFA (0.05% w/v) and immunoprecipitated with anti-HA antibody. Total cell lysates and precipitates were subjected to SDS-PAGE and analyzed by Western blotting for Vpu-HA, AP-1 Ɣ or AP-2 α. Panels are of representative experiments. Histograms represent quantification of the relative AP-1 or AP-2 binding normalized to input control. Error bars represent the standard deviation of three independent experiments.(TIF)Click here for additional data file.


Promyelocytic leukemia nuclear bodies (PML-NBs) are PML-based nuclear structures that regulate various cellular processes. SUMOylation, the process of covalently conjugating small ubiquitin-like modifiers (SUMOs), is required for both the formation and the disruption of PML-NBs. However, detailed mechanisms of how SUMOylation regulates these processes remain unknown. Here we report that SUMO5, a novel SUMO variant, mediates the growth and disruption of PML-NBs. PolySUMO5 conjugation of PML at lysine 160 facilitates recruitment of PML-NB components, which enlarges PML-NBs. SUMO5 also increases polySUMO2/3 conjugation of PML, resulting in RNF4-mediated disruption of PML-NBs. The acute promyelocytic leukemia oncoprotein PML-RARα blocks SUMO5 conjugation of PML, causing cytoplasmic displacement of PML and disruption of PML-NBs. Our work not only identifies a new member of the SUMO family but also reveals the mechanistic basis of the PML-NB life cycle in human cells. Promyelocytic leukemia nuclear bodies (PML-NBs) are non-membrane-bound domains in the cell nucleus that regulate transcription, antiviral response, DNA repair, apoptosis, senescence, and tumor suppression23Arsenic trioxide (ATO) induces the formation of PML-NBs through a two-step process: first, oxidized PML dimerizes using the BRCC domain710The recruitment of PML-NB components depends on SUMOylation212141516Similar to the formation of PML-NBs, disruption of PML-NBs also requires SUMOylation. Under oxidative stress, PML is conjugated by polySUMO2/3 chainsPML and RARα in APL patients. Although it is not clear how PML-RARα causes the disruption of PML-NBs, several clues suggest that SUMOylation of K160 was involved in this process: PML-RARα is a SUMOylated protein1415In addition to oxidative stress, PML-NBs can be disrupted by oncoprotein PML-RARα232425To understand how PML-NBs form in the absence of SUMO1, how K160 of PML controls the life cycle of PML-NBs, and how SUMOylation is involved in PML-RARα-mediated disruption of PML-NBs, we looked for other SUMO isoforms that could regulate PML-NBs. Here, we report the identification of SUMO5 that regulates the formation and disruption of PML-NBs in human cells. PolySUMO5 conjugation on K160 of PML results in recruitment of proteins to form PML-NBs. SUMO5 conjugation on PML is gradually replaced by SUMO2/3 conjugation, which leads to RNF4-mediated disruption of PML-NBs. Furthermore, PML-RARα causes disruption of PML-NBs by depriving PML of SUMO5 conjugation, leading to cytoplasmic displacement of PML. Our findings demonstrate that SUMO5 is a natural link controlling the dynamics of PML-NBs.SUMO1, we cloned a novel SUMO isoform we called SUMO5. The SUMO5 gene (GenBank: FJ042790.1) is located on chromosome 20 while SUMO1 (GenBank: NG_011679.1) is located on chromosome 2 (2q33). The cDNA of human SUMO5 contains a protein-coding sequence of 306 nucleotides, a 3′ noncoding region, and a poly(A) tail signal   databases, we found that SUMO5 was highly conserved among 17 primate species but more distantly related to the mouse ortholog (rom mice . SUMO5 ees (PBL) . In conte tissue . Moreoveg region , indicatTo determine if SUMO5 was capable of SUMO conjugation (SUMOylation), we tested if SUMO5 interacted with proteins involved in the SUMOylation process. SUMOylation requires the collaboration of SUMO E1, E2, and sometimes E3 enzymes, whereas attached SUMOs can be deconjugated by SUMO proteases, SENPs. Using an anti-Flag immunoaffinity purification strategy, we found that SAE2, a SUMO E1 enzyme, was present in the SUMO5 protein complex . Co-immuSequence analysis showed that SUMO5 contained a conserved SUMO modification motif IKDED (residues 17–21). This motif is absent in SUMO1 but corresponds in position to the SUMO modification motif of SUMO2/3  and S2B.Confocal microscopic analysis of cells expressing these mutant SUMO proteins  showed tAs SUMO5-induced NBs morphologically resembled PML-NBs, we hypothesized that SUMO5 regulates the formation of PML-NBs. Endogenous PML-NBs in cells were few and small A,B.To further distinguish the functions of SUMO5 and SUMO1 regarding the control of PML-NB formation, we created a cell-based reconstitution system by depleting both SUMO1 and SUMO5 using an shRNA construct and then adding back SUMO5 or SUMO1. The percentage of cells having detectable PML-NBs decreased from 73% to 54% when both SUMO1 and SUMO5 were knocked down C,D. HoweWe hypothesized that SUMO5 increased the size and number of PML-NBs through conjugation of PML. By detecting SUMO conjugation on PML using immunoprecipitation from cell lysates, we confirmed that PML-I, -IV and -VI, three PML isoforms we examined, were conjugated by SUMO5 . MoreoveNext, we examined whether different SUMO5 conjugation sites on PML were functionally distinct by using PML point mutants . PML(3K)SUMO5 contained an internal SUMO modification motif (IKDED) required for PML-NB formation . ThroughAlthough PML conjugated by wild-type SUMO5 appeared as smearing, conjugation by SUMO5(sm), SUMO5(K18R) or SUMO1 created distinct SUMOylated species of PML as sharp ladders (The major SUMO5 conjugation sites on PML (K160 and K490) overlapped the conjugation sites by SUMO1 reported in the literature. To further elucidate the differences between SUMO5 and SUMO1 regarding PML-NB formation, we performed SUMO conjugation assay and found that increasing amounts of SUMO1 enhanced conjugation of PML by SUMO5 but not vice versa  and S5A.To understand how increased SUMO5 conjugation to PML affected the formation of PML-NBs, we expressed SUMO5 and found that SUMO5 enlarged NBs containing SP100 and HIPK2  as well Although SUMO5 facilities the growth of PML-NBs, there was heterogeneity among NBs co-expressing SUMO2/3 and SUMO5 . To diff15We have shown that SUMO5 mediated the formation and normal turnover of PML-NBs , 4, 5, 6SUMO5 polyconjugates PML by successive self-conjugation at K18, and the internal SUMO modification motif (IKDED) of SUMO5 containing K18 is required for PML-NB formation  and 3. F121416Our results show that replacement of the SUMO5 conjugation of K160 on PML by SUMO2/3 mediates the disruption of PML-NBs. This transition from polySUMO5 conjugation to polySUMO2/3 conjugation recruits RNF4 to facilitate the disruption of PML-NBs. These data explain how PML-NBs disrupt under normal conditions, which resembles a previous PML-NB disruption model under oxidative stress10PML is conjugated by SUMO5 with a greater efficiency than PML-RARα when they are expressed individually . HoweverPrevious studies have reported that PML-NBs are stress-induced subnuclear structuresAlthough SUMO5 is highly homologous to SUMO1, it differs from SUMO1 in several remarkable aspects: First, it is conserved among primate species but appears not to be expressed in mice. Second, it is transcriptionally active in selective tissues, with high expression in testes and in blood cells. Theses attributes suggest that the mechanism regulating the life cycle of PML-NBs in human might differ from that in mice. However, despite persistent effort, we were unable to produce an antibody specific for SUMO5 due to the extremely high degree of identity between SUMO5 and SUMO1. Nevertheless, by using shRNA specifically targeting SUMO5, we showed SUMO5 was a critical regulator of the PML-NB cycle. Our study, which is the first comprehensive analysis of this novel member of the SUMO family, provides critical mechanistic details about the life cycle of PML-NBs.SUMO5 gene, we found that the putative SUMO5 promoter contains two C/EBP-β bindings sites and nine GATA-1 binding sites among other transcription factor binding motifs. This information suggests that SUMO5 is potentially under the regulation of multiple transcription factors in normal, physiologic conditions, with C/EBP-β and GATA-1 being the most prominent regulators of SUMO5 expression. C/EBP-β is critical for the differentiation of adipocytes42Through bioinformatic interrogation of the sequence upstream from the SUMO5 is controlled by transcription factors including C/EBP-β, whose own expression might also change when normal cell physiology is perturbed. As a result, the abnormal pattern of SUMO5 conjugation on target proteins such as PML and PML-RARα will likely to be reinforced in APL.Our analysis on how PML-RARα highjacks SUMO5 conjugation demonstrates that the control of SUMO5 conjugation is important in the pathogenesis of APL. Our data also show that SUMO5 is abundantly expressed in the APL cell line NB4 . InteresFJ042790.1) was specifically selected based on the formation of NBs.SUMO5 cDNA was identified by RT-PCR using primers based on the coding region of SUMO1 cDNA and a cDNA library prepared from total RNA of 293 cells. Subsequently, PCR products were cloned in-frame into a GFP fusion expression vector (pEGFP-C1). Each clone was transfected into 293 cells. SUMO5 (GenBank accession number: M73778.1) and PML(3K) were kindly provided by Dr. Hsiu-Ming Shih. Flag-PML-I, Flag-PML-IV, and Flag-PML-VI were kindly provided by Dr. Ruey-Hwa Chen. Plasmids expressing Flag-, HA-, GFP-tagged PML and PML(3K) proteins and plasmids expressing Flag- or HA-tagged Daxx proteins were obtained by subcloning into pcDNA3-Flag, pcDNA3.1-HA, and pEGFP-C1 vectors. Plasmids expressing Flag-tagged SP100 and HIPK2 were provided by Dr. M. Lienhard Schmitz. Flag-tagged SENP1 was kindly provided by Dr. Kung-Yao Chang. Plasmids expressing HA-tagged Ubc9, PIAS1, and PIASx-α were kindly provided by Dr. Keith D. Robertson. Site-directed mutagenesis was performed to obtain the following mutants: SUMO5(H12D), SUMO5(K18R), SUMO5(I95T), SUMO5(sm), SUMO1(IKDE), SUMO1(IKDΔ), SUMO5(I95R), PML(K65R), PML(K160R), PML(K380R), PML(K400R), PML(K490R), PML(K497R), and Ubc9(DN)(C93S). pGEX-2TK-hPc2, the plasmid expressing GST-tagged Pc2, was kindly provided by Dr. David P. E. Satijn. HA-tagged Pc2 was obtained by insertion of a PCR-amplified DNA fragment of Pc2 into pcDNA3.1-HA vector. Plasmids expressing Flag-tagged RNF4 and its mutant RNF4(3CS) were kind gifts of Dr. Ronald T. Hay.Coding regions of SUMO5, SUMO1, SUMO2, SUMO3, and SUMO4 were amplified from 293 cells by RT-PCR and then inserted into pEGFP-C1 (Clontech) to create plasmids expressing GFP-tagged SUMO proteins. cDNA fragments of SUMO5, SUMO1, and SUMO3 were subcloned from pEGFP-C1 into pDsRed1-C1 (Clontech), pmCherry-C (Clontech), pcDNA3.1-HA, and pcDNA3-Flag vectors to obtain plasmids expressing RFP-, mCherry-, HA-, and Flag-tagged proteins. The plasmid expressing GFP-tagged SUMO5(AA) was prepared using site-directed mutagenesis  and the fragment of SUMO5(AA) was further subcloned into pcDNA3.1-HA and pcDNA3-Flag vectors. The coding region of ubiquitin was amplified from 293 cells by RT-PCR and then inserted into pcDNA3-Flag to create the plasmid expressing Flag-tagged ubiquitin. GFP-tagged Daxx, HA-tagged PML-RARα, and cDNAs of PML , HeLa (human cervix adenocarcinoma), MCF7 (human breast adenocarcinoma), A2058 (human metastatic melanoma), K562 (human chronic myelogenous leukemia), NIH 3T3 (mouse embryonic fibroblast), and B16F10 (mouse skin melanoma) cells. NB4 (human acute promyelocytic leukemia) cells were grown in RPMI with 20% fetal bovine serum and 1% penicillin-streptomycin. Expression plasmids were transfected into 293 cells using a calcium phosphate co-precipitation method. N-ethylmaleimide , dissolved in 95% ethanol and stored at a concentration of 0.4 M at −20 °C, was used at a concentration of 10 mM by adding into the cell lysis buffer.For the analysis of SUMO5 expression in cell lines, total RNA was isolated from 293, K562, NB4, A2058, MCF7, HeLa, B16F10, and NIH 3T3 cells using a WelPrep Cell/Tissue RNA Kit (Welgene). Extracted RNAs were used as template in reverse transcriptase PCR with oligo-dT primers and NxGen™ M-MuLV Reverse Transcriptase (Lucigen), and followed by PCR with the SUMO5-specific primer pair. For the analysis of SUMO5 expression in tissues, cDNAs from Multiple Tissue cDNA Panels Human I and II (Clontech) were used as template for PCR. GAPDH was used as internal control in Small hairpin RNA (shRNA) constructs shSUMO1/5 (RHS4430-98481086), shUbc9 (RHS4430-98520611), shSUMO5 (customized SUMO5-specific shRNA) and shNC (negative control shRNA) were obtained from GenDiscovery Biotechnology. The target sequences of shSUMO1/5, shUbc9, shSUMO5, and shNC are TTATCAGCAATTCTCTGAC, TTAGTGAAGCCACAGATGTAA, GGGATAAGATAAAAGATGAAG, and TTCTCCGAACGTGTCACGT, respectively. shRNA was delivered into cells by calcium phosphate co-precipitation. In the rescue assay, shSUMO1/5 was first transfected into 293 cells. 24 hours later, the culture media were discarded and cells were washed three times with PBS prior to the second transfection with the SUMO5 or SUMO1 expression construct.Mouse monoclonal anti-HA (H9658) and anti-FLAG M2 (F1804) antibodies were from Sigma-Aldrich. Mouse monoclonal anti-SUMO1 (sc-5308), mouse monoclonal anti-hPML (sc-966), goat polyclonal anti-hSP100 (sc-16328), mouse monoclonal anti-gamma-tubulin (sc-17788), mouse anti-β-actin (sc-47778), and rabbit polyclonal anti-hRAR-α (sc-551) antibodies were from Santa Cruz Biotechnology. Mouse anti-KAP1 antibody was made in house. Rabbit polyclonal anti-GFP antibody was a kind gift from Dr. Liang-Jwu Chen. Mouse anti-GFP monoclonal antibody clone 3D8A1B8 (Gm0001-02) was from Abking Biotechnologies Inc.To perform serial IP, anti-Flag IP was carried out first using standard procedures. After washing, immunoprecipitated proteins were eluted by adding 300 μl of PBS containing 0.1% NP-40 and 100 μg/ml of a Flag peptide  for 30 min on ice. After pulse centrifugation , the soluble phase was collected for anti-HA IP.293 cells were grown on glass coverslips in 6 cm dishes and transfected as described. After 24 hours, cells were treated with reagents for the time indicated and fixed with 3% paraformaldehyde for 10 min at room temperature at indicated time points. Fixed cells were washed with PBS once and permeabilized with 0.2% Triton X-100 in PBS. For immunolocalization of endogenous proteins, cells were incubated with primary antibodies and fluorescently labeled secondary antibodies. Fluorescence microscopy analysis was carried out using an Olympus A BX51 upright epi-fluorescence microscope with an UplanFL 100× objective. Confocal microscopy analysis was carried out with an Olympus FluoView FV1000 laser-scanning microscope using a 63 × 1.4 numerical aperture objective.8 293 cells expressing Flag-SUMO5 were lysed in PBS containing 0.1% NP-40 followed by centrifugation at 12,000 rpm for 10 min to obtain a cleared lysate. The lysate was applied to a gravity column (Bio-Rad Laboratories) containing 600 μl of anti-Flag M2-Agarose  and re-applied for a total of three times. The affinity column was washed extensively with the lysis buffer, and the bound SUMO5 complex was eluted from the column with 3 ml of an elution buffer containing 20 μg/ml of Flag peptide . Five fractions of eluate were obtained, and the third fraction was air-dried to concentrate to the proper volume before SDS-PAGE separation using 3% stacking/12% resolving polyacrylamide gels. Protein bands were stained with silver for visualization.Approximately 1.7 × 10TM well-plate microautosampler. A full-mass scan was performed between m/z 350 and 2000, followed by MS/MS scans of the five highest-intensity parent ions at 35% relative collision energy. The AGC of fluoranthene anions was set at 3 × 105 and the reaction time was set at 200 ms during ETD.In-gel digestion of selected protein bands was performed essentially as describedHomo sapiens  was chosen as the taxonomic category. For the identification of SUMO5 modification sites on PML, the acquired MS/MS spectra were searched against Swiss-Prot (released 27 August 2013) using Mascot Daemon version 2.3.2. Mammalia  was chosen as the taxonomic category. Masses for both precursor and fragment ions were treated as monoisotopic. In addition, an in-house database containing SUMO5 (GI: 52783791), SUMO1 (GI: 52783799), PML-IV , and Pc2 (GI: 55770830) was used.For the identification of proteins in the SUMO5 complex, the acquired MS/MS spectra were searched against the Swiss-Prot protein database (released 8 January 2008) using Mascot Daemon version 2.2.2. Accession codes: The SUMO5 gene (GenBank: FJ042790.1); SUMO1 gene (GenBank: NG_011679.1); SUMO1 protein (GenBank: AAC50996.1); SUMO2 protein (GenBank: AAH71645.1); SUMO3 protein (NCBI Reference Sequence: NP_008867.2); SUMO4 protein (NCBI Reference Sequence: NP_001002255.1); Homo sapiens promyelocytic leukemia (PML), transcript variant 6 . In Fig. 1B, the sequence accession numbers of SUMO5 orthologs are: Human , Chimp , Bonobo , Gorilla , Orangutan , Gibbon , Rhesus , Crab-eating macaque , Baboon , Green monkey , Proboscis monkey , Golden snub-nosed monkey , Marmoset , Squirrel monkey , Tarsier , Mouse lemur , Bushbaby , Tree shrew , Mouse , Dog .How to cite this article: Liang, Y.-C. et al. SUMO5, a Novel Poly-SUMO Isoform, Regulates PML Nuclear Bodies. Sci. Rep.6, 26509; doi: 10.1038/srep26509 (2016).


Drosophila, the PTEN ortholog and its mammalian counterpart downregulate insulin/IGF signaling by antagonizing the PI3-kinase function. From a dominant loss-of-function genetic screen, we discovered that mutations of a Dbl-family member, the guanine nucleotide exchange factor DRhoGEF2 (DRhoGEF22(l)04291), suppressed the PTEN-overexpression eye phenotype. dAkt/dPKB phosphorylation, a measure of PI3K signaling pathway activation, increased in the eye discs from the heterozygous DRhoGEF2 wandering third instar larvae. Overexpression of DRhoGEF2, and it’s functional mammalian ortholog PDZ-RhoGEF (ArhGEF11), at various stages of eye development, resulted in both dPKB/Akt-dependent and -independent phenotypes, reflecting the complexity in the crosstalk between PI3K and Rho signaling in Drosophila.The insulin/IGF-1 signaling pathway mediates various physiological processes associated with human health. Components of this pathway are highly conserved throughout eukaryotic evolution. In  In higher eukaryotes, the Insulin/IGF-1 signaling pathway plays a key role in control of growth, development and differentiation, metabolic homeostasis and aging, acting via the insulin receptor (IR) and the insulin-like growth factor receptor (IGF-1R)  Briefly,Drosophila, a PTEN ortholog and its mammalian counterpart negatively regulate insulin/IGF signaling by antagonizing PI3-kinase function. PTEN (phosphatase and tensin homology on chromosome 10) is frequently deleted in advanced human cancers. Germ line loss of PTEN is directly linked to the development of the PTEN hamartoma tumor syndrome (PHTS), a predisposition for the development of benign tumors in various organs [The components of this pathway are highly conserved throughout eukaryotic evolution. In s organs . Somatics organs . Moreoves organs . It is ws organs , 17. PIPs organs , 19. By s organs , 21. Acts organs , 22–25. s organs –28. PTENs organs –29.Drosophila compound eye and various tissues in mice [Drosophila compound eye.Conserved PTEN function has been characterized in a tissue-specific or cell-type specific fashion in both  in mice , 30. We  in mice , 32. DRhw+;GMR-GAL4>UAS-PTEN/CyO) was generated in our lab as described previously [P-element line for DRhoGEF2 , the driver lines, GMR-GAL4/II, EYE-GAL4/II, and EMS (Ethylmethanesulfonate) Rho1 mutant line (Rho1E3.10),  [Drosophila Rho kinase mutant line, Drok2  [P-element enhancer line of RhoGAPp190 (RhoGAPp190EY08765)  and several RNAi mutant lines were obtained from Bloomington Drosophila Stock Center at Indiana University: GFPRNAi , DRhoGEF2RNAi , and Rho1RNAi . Two EMS-induced mutant lines DRhoGEF23w18/CyO and DRhoGEF24.1/CyO were kindly provided by Dr. Armen Manoukian, Department of Medical Biophysics, University of Toronto  [w+;+/+;ry506, and w1118 were used as wild type controls. Stocks were maintained and all experiments were conducted at 25°C on a 12h:12h light:dark cycle at constant humidity using standard sugar/yeast/agar (SYA) medium.The PTEN overexpression transgenic fly line (eviously . The P-eer 3167) , Drosopher 6665) , 36, andDRhoGEF2 was cloned from a Drosophila melanogaster BAC clone containing DRhoGEF2 cDNA obtained from Research Genetics, subcloned into pUAST [pUAST-DRhoGEF2, pUAST-PDZ-RhoGEF, and pUAST-PDZ-RhoGEFd8 transgenenic line by injection into w1118 embryos for germ line transformation as described previously [DRhoGEF2 transgenic lines were generated .The 8.6 kb full-length DNA of to pUAST  and usedeviously . Three Dw+;UAS-DRhoGEF2/CyO or w+;UAS-mycPDZ-RhoGEF/CyO in the fly in the presence of a P-element insertion mutant allele of DRhoGEF2, w+;DRhoGEF204291/CyO and chemically induced point mutation w+;DRhoGEF23w18/CyO. Virgin females carrying w+;DRhoGEF204291/CyO;ARM-GAL4/TM3 were crossed to males carrying w+;DRhoGEF23w18/CyO;UAS-DRhoGEF2wt/TM3, w+;DRhoGEF23w18/CyO;UAS-mycPDZ-RhoGEF/TM3, or w+;DRhoGEF23w18/CyO;UAS-mycPDZ-RhoGEFd8/TM3. The genotype of F1 flies; w+;DRhoGEF204291/DRhoGEF23w18;ARM-GAL4/UAS-DRhoGEF2wt, w+;DRhoGEF204291/DRhoGEF23w18;ARM-GAL4/UAS-mycPDZ-RhoGEF, and w+;DRhoGEF204291/DRhoGEF23w18;ARM-GAL4/UAS-flagPDZ-RhoGEFd8 were assayed for viable adult flies. At least 2,000 flies were scored.A ARM-GAL4 binary system was used to express transgene: GMR-GAL4 (glass multiple reporter driven GAL4 expression) was employed to drive expression in the R cells in the eye imaginal disc and ey-GAL4 (eyeless promoter driven GAL4 expression) was used to overexpress the transgenes in the anterior, undifferentiated region of the eye imaginal disc during the third instar larval stage [Standard genetic crosses were set up for ectopic expression of DRhoGEF2 in the fly eyes. DRhoGEF2 was overexpressed in the specific stage of eye development using the upstream activation sequence (UAS)-GAL4 binary system . During al stage .Drosophila eyes were fixed in 2% osmium/1% glutaraldehyde/0.1 M phosphate buffer (pH 7.2) for 30 min and followed by one change with fresh 2% osmium. After washing with 0.1 M phosphate buffer, osmiums fixed eyes were dehydrated with ethanol and ethanol was replaced by propylene oxide. Eyes were embedded in Durcapan resin mixture  in the modules for sectioning. Sections were stained with 1% toluidine blue solution.hsflp;act>y+>GAL4UASGFP/CyO were crossed with w+;UAS-DRhoGEF2/UAS-DRhoGEF2 or w+;UAS-mycPDZ-RhoGEF/UAS-mycPDZ-RhoGEF at 18°C for 3 days, then, parental flies were flipped out. Embryos were heat shocked for 45 min at 37°C and maintained at 25°C. Eye imaginal discs from wandering third-instar larvae were dissected and fixed in PBS/4% PFA (Sigma-Aldrich), washed in PBS/0.1% Triton X-100 (Sigma-Aldrich), and incubated overnight with primary antibody. Discs were stained with rat anti-Elav , goat-anti-rat-Cy5 (Jackson Lab), and phalloidin-rhodamine (Molecular Probe). The stained discs were analyzed with a Zeiss confocal microscope.The eye imaginal discs were dissected from the third-instar larvae in S2 insect medium. Apoptosis was determined by staining with 3 mg/ml of acridine orange (Sigma-Aldrich). For cell proliferation, dissected discs were labeled with BrdU  as described . BrieflyAll adult eye phenotypes were analyzed in females raised at 25°C unless indicated otherwise. The external eye phenotype was analyzed using a standard protocol for scanning electronic microscopy. For ommatidial organization, transverse sections were prepared for light and transmission electron microscopy.To prepare total protein lysates, five to 10 eye imaginal discs were homogenized in cell lysis buffer (20 mM Tris (pH7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton x-100, phosphatase inhibitors , and protease inhibitors ). Phosphorylation of dPKB/dAkt (serine 505), total dPKB/dAkt, and β-tubulin were detected using antibodies for phospho-S505 of dPKB/dAkt and total dPKB/dAkt , β-tubulin (Upstate).PTEN expression to a collection of 1045 P-element strains. Each strain comprises a single P-element insertion in one allele of each gene, which when homozygous leads to embryonic lethality [P-element insertions, I(2)04291, which maps to 53F01-2 cytological location on the right arm of chromosome 2, partially rescued the PTEN-driven small eye phenotype (I(2)04291 inserts at the 5’-end of the promoter region of DRhoGEF2 and disrupts its expression (DRhoGEF204291) [P-element insertion line in the same gene, DRhoGEF2e03784 042F204291) . The intF2e03784  and DRhoon group , 32  (Rho1 RNAi (Rho1RNAi)   or upon ho1RNAi) .DRhoGEF204291 and GMRGAL4>DRhoGEF2RNAi flies (Rho1 (Rho1E3.10) and Drok (Drok1), the downstream effectors of DRhoGEF2, also displayed elevated dPKB/dAkt S505 phosphorylation , under the control of the armadillo-GAL4 (ARM-GAL4) system driving transgene expression during early embryo development, rescued the lethality caused by the homozygous DRhoGEF204291 (DRhoGEF204291/DRhoGEF204291) or the heterozygous DRhoGEF204291 with the EMS allele DRhoGEF23w18 (DRhoGEF204291/DRhoGEF23w18)  . Of noteardation , resultiardation .DRhoGEF2 or PDZ-RhoGEF was placed under the control of eyeless-GAL4 (ey-GAL4), resulting in expression in the neuronal precursor cells at the anterior of the morphorgentic furrow (MF). DRhoGEF2 overexpression led to severe eye damage, small or no eye phenotype , which transmit Gɑ-protein coupled receptor (Fog/Cta)-dependent and -independent signals to Rho1, to regulate cell shape, invagination, and epithelial folding during embryogenesis and eye development [DRhoGEF2 and the Drosophila effector Rho1, genetically interact with PTEN. DRhoGEF2 loss of function increases dPKB/dAkt activity and suppresses the eye phenotype elicited by PTEN-overexpression, further connecting the Rho1 and PI3K pathways in the Drosophila eye. Importantly, DRhoGEF2 and human PDZ-RhoGEF are functionally redundant in maintaining ommatidia integrity.DRhoGEF2 is a elopment , 44–46. DRhoGEF204291 and RhoE3.10 eye discs with reduced Rho signaling , chico (Drosophila Insulin Receptor Substrate (IRS)), PI3-kinase, and dPKB/dAkt, lead to reduced cell growth during Drosophila eye and wing development –50 and in clones . Analogon clones . Our finn clones , 54. Ourn clones , 55, 56,rd instar larval eye imaginal eye discs  GMR-GAL4>UAS-PTEN/DRhoGEF23w18, and (III) GMR-GAL4>UAS-PTEN/+;GFPRNAi/+, (IV) GMRGAL4>UAS-PTEN;DRhoGEF2RNAi/+. Scale bar = 200 μm. (B) The ommatidial number in individual flies was determined by scanning electronic micrographs (n = 10). (C) The levels of dPKB/dAkt phosphorylation in the 3rd instar larval eye imaginal discs in GMRGAL4/+;GFPRNAi/+ and GMRGAL4/+;Rho1RNAi/+ and quantified using ImageJ. (D) Scanning electronic micrographs of adult fly eyes from (I) GMRGAL4>UAS-PTEN;GFPRNAi/+ and (II) GMRGAL4>UAS-PTEN/+;DRhoGEF2RNAi/+. Scale bar = 200μm.(A) Scanning electronic micrographs of adult eyes from (I) (TIF)Click here for additional data file.S2 FigARMGAL4 driven DRhoGEF2 or PDZ-RhoGEF overexpression exhibited growth retardation and died during late 2nd or early 3rd instar larval stage.(A) An unrooted phylogenetic analysis based on the ClustlW alignment of the amino acid sequence of five members of RGS-RhoGEF subfamily. The phylogenetic tree demonstrated that PDZ-RhoGEF is the closest mammalian ortholog of DRhoGEF2. (B) Embryos with (TIF)Click here for additional data file.S3 FigDRhoGEF2 or mycPDZ-RhoGEF under the control of GMR-GAL4. (I) GMR-GAL4/+, (II) GMR-GAL4/UAS-DRhoGEF2, and (III) GMR-GAL4/UAS-mycPDZ-RhoGEF. Scale bar = 200 μm. (B) Toluidine blue-stained transverse sections of the adult eye with DRhoGEF2 or PDZ-RhoGEF overexpression. (I) GMR-GAL4/+, (II) GMR-GAL4/UAS-DRhoGEF2, and (III) GMR-GAL4/UAS-mycPDZ-RhoGEF. (C) Acridine orange (AO) staining in the 3rd instar larval eye imaginal discs with DRhoGEF2 or mycPDZ-RhoGEF overexpression. (I) GMR-GAL4/+, (II) GMR-GAL4/UAS-DRhoGEF2, and (III) GMR-GAL4/UAS-mycPDZ-RhoGEF. (D) Cell proliferation in DRhoGEF2- or PDZ-RhoGEF-overexpressing 3rd instar larval eye imaginal discs, determined by BrdU incorporation. (I) GMR-GAL4/+, (II) GMR-GAL4/UAS-DRhoGEF2, and (III) GMR-GAL4/UAS-mycPDZ-RhoGEF. (E) & (F) Phosphorylation of dPKB/dAkt in the 3rd instar larval eye imaginal discs with DRhoGEF2 (C) or PDZ-RhoGEF (D) overexpression.(A) Scanning electron micrographs of adult eye s with ectopic expression of (TIF)Click here for additional data file.S4 FigDRhoGEF2 (A) or PDZ-RhoGEF (B) expression induced by heat shock through mitotic recombination. (C) Scanning electronic micrographs of adult eyes from heat-induced recombination and gene expression. (I) hsflp;act,FRT,GAL4>UAS-GFP/UAS-DRhoGEF2 and (II) hsflp;act,FRT,GAL4>UAS-GFP/UAS-mycPDZ-RhoGEF. (D) Scanning electronic micrographs of adult fly eyes from GMR-GAL4>UAS-DRhoGEF2/CyO (I), GMR-GAL4>UAS-DRhoGEF2/RhoE3.10 (II), w67c23P{EPgy2}RhoGAPp190EY08765/+;GMR-GAL4>UAS-DRhoGEF2 (III). Scale bar = 200 μm.(A) & (B) Immunostaining of the post-mitotic neuronal cells with ectopic (TIF)Click here for additional data file.


Smurf2 had an expanded hematopoietic stem cell (HSC) compartment in bone marrow under normal homeostatic conditions, and this expansion was associated with enhanced proliferation and reduced quiescence of HSCs. Surprisingly, increased cycling and reduced quiescence of HSCs in Smurf2-deficient mice did not lead to premature exhaustion of stem cells. Instead, HSCs in aged Smurf2-deficient mice had a significantly better repopulating capacity than aged wild-type HSCs, suggesting that decline in HSC function with age is Smurf2 dependent. Furthermore, Smurf2-deficient HSCs exhibited elevated long-term self-renewal capacity and diminished exhaustion in serial transplantation. As we found that the expression of Smurf2 was increased with age and in response to regenerative stress during serial transplantation, our findings suggest that Smurf2 plays an important role in regulating HSC self-renewal and aging.The age-dependent decline in the self-renewal capacity of stem cells plays a critical role in aging, but the precise mechanisms underlying this decline are not well understood. By limiting proliferative capacity, senescence is thought to play an important role in age-dependent decline of stem cell self-renewal, although direct evidence supporting this hypothesis is largely lacking. We have previously identified the E3 ubiquitin ligase Smurf2 as a critical regulator of senescence. In this study, we found that mice deficient in  Tissue-specific stem cells, which are capable of self-renewal to preserve stem cell pools as well as differentiation into a variety of effector cells, play a central role in the normal homeostatic maintenance and regenerative repair of tissues throughout the lifetime of an organism. With advancing age, the self-renewal capacity of stem cells invariably declines, eventually leading to the accumulation of unrepaired, damaged tissues in old organisms  and p53 have been found to play important roles in aging. The expression of p16 increases with age in many human and rodent tissues  and the LSK  population that is enriched for HSCs  C57BL/6 mice compared with young (2-month) mice , in which a gene-trapping cassette is inserted into intron 1 of Smurf2 to disrupt its normal splicing  mice , short-term HSCs (ST-HSCs: Lin−Sca1+c-kit++CD150−Flt3−), and multipotent progenitors (MPPs: Lin−Sca1+c-kit++CD150−Flt3+)  in the total live BM cells collected from long bones of hind and forelegs of 2-month-old Smurf2-deficient mice compared with age-matched WT mice  compared with WT mice   compared with WT mice  mice, we also found an increase in BM cellularity in Smurf2-deficient mice  or old (18–20 months) age in various lineages, including white blood cells, red blood cells, lymphocytes, granulocytes, monocytes, and platelets Fig. . These rP = 0.0012). To further investigate whether cell proliferation is enhanced in Smurf2-deficient LT-HSCs and progenitor populations, we gave young mice 4 injections of bromodeoxyuridine (BrdU) in a span of 24 h to label LT-HSCs  using this labeling method  compared with WT LT-HSCs  populations compared with WT cells  exhibited decreased frequency in the G0 phase , and increased frequencies in the G1  and S-G2/M phases   populations also showed significantly increased cycling and decreased quiescence  were mixed at a 1:1 ratio with competitor BM cells of 2-month-old CD45.1+/CD45.2+ WT mice and injected into lethally irradiated congenic recipient mice (CD45.1+). Contribution of donor, competitor, and recipient in peripheral blood and BM of reconstituted recipients was determined by flow cytometry at different time post-transplantation. As we have found previously that ~30% of Smurf2-deficient mice develop tumor spontaneously after 12 month of age, and ~70% of tumors are B-cell lymphomas in spleen , suggesting that Smurf2-deficient HSCs have an enhanced long-term self-renewal capacity. None of these recipient mice died during 1 year of observation. We carried out a 5th transplantation with Smurf2-deficient donors and found that 60% of the recipient mice survived for 1 year post-transplantation without signs of illness or stress . Collectively, these results indicate that Smurf2-deficient LT-HSCs have a better capacity in the long-term self-renewal and multilineage potential than the WT LT-HSCs. Furthermore, this stem cell-intrinsic functional enhancement is maintained in aged Smurf2-deficient mice.Similarly, we carried out BM serial transplantation using aged mice as donors. All recipients receiving donor BM from either aged WT or Smurf2-deficient mice survived during the first 3 cycles of transplantation. Complete blood count analysis of recipient peripheral blood 3 months after transplantation showed that various blood cell lineages were successfully reconstituted by both the WT and Smurf2-deficient donors Fig. . During p18INK4c (p18) have increased self-renewal ability accompanied by increased cycling , which are located in the same INK4a locus, was increased in BM of aged (24-month) WT C57BL/6 mice compared with young (2-month) mice  for more than 10 generations. All mouse studies were carried out according to guidelines approved by the Institutional Animal Care and Use Committee of University of Massachusetts Medical School.Smurf2-deficient mice as described previously .-1 propidium iodide to exclude dead cells. Flow cytometry analysis was performed on a 5-laser, 18-detector LSR II FACS , and data were analyzed using FlowJo software .Bone marrow cells were harvested from long leg bones and resuspended in staining medium consisting of biotin-, flavin-, and phenol red-deficient RPMI 1640 medium  supplemented with 2% FBS , 10 mM HEPES (pH 7.2), 1 mM EDTA, and 0.02% sodium azide. After filtering through 70-μm nylon mesh, BM cells were incubated with anti-CD16/32 antibody  for 10 min on ice to block Fc receptors and then incubated with primary antibodies for 20 min. The lineage cocktail contained biotin-conjugated antibodies to B220 (RA3-6B2), CD3e (145-2C11), CD11b (M1/70), CD19 (1D3), Ly-6G (RB6-8C5), and Ter-119 (TER-119). Additional antibodies for HSC analysis were Ly-6A/E(Sca1)-FITC or Ly-6A/E(Sca1)-PerCp-Cy5.5 (D7), CD117(c-Kit)-PE-Cy7 or CD117(c-Kit)-APC (2B8), CD135(Flt3)-PE (A2F10), and CD150-APC (mShad150). All antibodies were purchased from eBioscience . Cells stained with biotin-labeled antibodies were incubated with streptavidin-eFluor 450 (eBioscience) for 15 min on ice and washed three times with staining medium. After the final wash, cells were resuspended in staining medium with 1 μg mLet al., et al., Mice were injected with 1 mg BrdU intraperitoneally every 6 h for 24 h as described previously (Passegue +) were used as donors, and congenic CD45.1+ mice  were used as recipients. Eight- to ten-week-old recipient mice were lethally irradiated (10 Gy) using a Cs137 irradiator 24 h before transplantation and were treated with antibiotics (0.5 mg mL-1 neomycin and 100 U mL-1 polymyxin-B) in drinking water 24 h prior to exposure to radiation until 1 month after transplantation. For serial transplantation, 5 × 106 donor BM cells were injected retro-orbitally into 8- to 10-week-old lethally irradiated recipient mice. The transplantation cycle was repeated every 2 months.Wild-type or Smurf2-deficient mice  and CD45.1-PE-Cy7 (A20). Multilineage reconstitution in peripheral blood was analyzed using antibodies to B220-APC (RA3-6B2), CD3e-biotin (145-2C11), and CD11b-APC-Cy7 . Antibodies were purchased from eBioscience unless specified.For competitive repopulation, 2-month-old WT CD45.1Total RNA was isolated from freshly collected BM or FACS-sorted LSK cells using TRIzol (Invitrogen) and reverse-transcribed using Superscript II reverse transcriptase (Invitrogen). Real-time PCR was carried out on MyiQ iCycler using SYBR Green PCR kit . The following primers were used: Smurf2 , p16 , p18 , p19 , and β-actin .t-test was used for pairwise comparisons. Two-way anova was used for multiple comparisons. Kaplan–Meier survival curves were plotted using GraphPad Prism 5.0, and statistical significance was analyzed using the log-rank test. P < 0.05 was considered as statistically significant.Data were presented as mean ± SD. Two-tailed and unpaired Student’s


While only 10-30% of patients treated with currently approved single immunotherapies demonstrate long-term benefit, treatment efficacy in responders correlates with increased CD8+ T cell responses to neoantigens (tumor antigens hidden from the immune system). Thus, to improve presentation of tumor antigens and expose neoantigens we engineered a single-step nanoparticle antigen presentation system (SNAPS) that enables both membrane and cytosolic cancer proteins to be covalently coupled to an injectable nanoparticle emulsion.B16 melanoma (SNAPS-B16), primary melanocyte (SNAPS-MEL), 4T1 breast cancer (SNAPS-4T1) lysate or mouse IgG (SNAPS-C) were used for coating polystyrene nanoparticles via an EDAC linker. One day or 11 days post B16-F10 tumor cell challenge , mice were treated (at the same site as the tumor cell injection) with 200 μL of 1% w/v cell lysate coated nanoparticles. Tumors were measured using calipers every 2-3 days. Flow cytometry staining was performed for antigen presenting cells (APCs) and CD8+ T cells after dissection and mechanical dissociation of the tumor. Statistical analyses were performed using the student t test and Prism v4.0 software (GraphPad). Differences with a P < 0.05 were considered statistically significant.in vivo, demonstrating the induction of an antigen-specific anti-tumor response with SNAPS-B16. Additionally, SNAPS-B16 significantly increased (P < 0.01) the proportion of APCs within the tumor microenvironment compared to the SNAPS-C, as evidenced by the increased percentage of MHC-II+ cells).We observed that SNAPS-B16 delivered at one day post tumor challenge significantly reduced tumor growth (P < 0.01) compared to SNAPS-C (which had no effect). SNAPS delivered at day 11 (when tumors were approximately 5 mm × 5 mm) significantly halted tumor growth (< 2-fold increase over the course of 10 days) while B16 treated with SNAPS-C increased ~16-fold (P < 0.01). Neither SNAPS-4T1 nor SNAPS-MEL had any effect on the growth of B16 in vivo may sidestep many of the complexities and per patient costs associated with current cell-based immunotherapies and tumor antigen sequencing.Our results demonstrate that SNAPS coupling of whole tumor lysate from cancer cells to nanoparticles delivers a robust anti-tumor immune response resulting in enhanced tumor regression. These findings demonstrate that a cancer-derived protein unique to B16 melanoma  coating the SNAPS confers tumor specificity. Although preliminary, our reported innovation of using nanoparticles coated through EDAC with tumor proteins in this specific manner to elicit immune responses against tumor cells


Assessing the extracellular volume fraction (ECV) by T1-mapping cardiovascular magnetic resonance (CMR) is an attractive approach to quantify myocardial injury as a potential predictor of adverse events in these patients. This study evaluated ECV imaging by T1-mapping CMR in comparison with the established "Seattle Heart Failure Model"  ejection fraction due to non-ischemic cardiomyopathy. The SHFM was used to estimate 1, 2 and 5 year mortalities as well as life expectancy, respectively. T1 quantification was performed at 1.5 Tesla using the modified Look-Locker inversion-recovery (MOLLI) sequence on 3 short-axes before and 15 minutes after administration of 0.075 mmol/kg gadolinium-BOPTA. Global myocardial ECV was then calculated from native and post-contrast T1 maps generated by a dedicated plug-in written for the OsiriX software, respectively.Median predicted 1-, 2- and 5-year mortalities were 4% (interquartile range 2-6%), 7% (interquartile range 5-12%) and 16% (interquartile range 12-28%) in the study population, respectively. Median estimated life expectancy was 13 years (interquartile range 9-15 years). A significant correlation was found between global myocardial ECV and predicted 1- , 2-  and 5-year mortality , respectively. Furthermore, a significant inverse correlation was found between global myocardial ECV and predicted life expectancy .Myocardial injury as quantified by ECV-imaging correlates well with predicted mortality and has great potential to improve risk stratification in patients with heart failure due to NIDCM.No funding.


Eratum: Recurrent ZFX mutations in human sporadic parathyroid adenomas.Chen-Pang Soong, Andrew Arnold360-366.(2014).Oncoscience 1: PMCID: PMC4278311. PMID: 25594030In Figures 1 and 2, the 3-letter amino acid code labels Glu should have been Gln. The corrected Figures 1 and 2 are provided here.


Atg9 is a multispanning transmembrane protein that is required for autophagosome formation. During autophagy, vesicles containing Atg9 are generated through an unknown mechanism and delivered to the autophagosome formation sites. We have previously reported that Atg9-containing membranes undergo continuous tubulation and fission during nutrient starvation in a manner dependent on the curvature-inducing protein Bif-1/Sh3glb1. Here, we identify Dynamin 2 (DNM2) as a Bif-1-interacting protein that mediates the fission of Atg9-containing membranes during autophagy. The interaction of Bif-1 and DNM2 is enhanced upon nutrient starvation, and Bif-1 and DNM2 cooperatively induce the generation of Atg9-containing vesicles. Inhibition of the GTPase activity of DNM2 results in the accumulation of Atg9-positive tubular structures that originate from a Rab11-positive reservoir. Although Atg9 seems to be constitutively trafficked to the reservoir regardless of Bif-1 expression, membrane tubulation from the Atg9 reservoir is dependent on Bif-1 and is strongly induced upon nutrient starvation. These findings suggest that the generation of Atg9 vesicles from a Rab11-positive reservoir is tightly controlled by the Bif-1-DNM2 membrane fission machinery in response to cellular demand for autophagy. Autophagy is an evolutionarily conserved lysosomal catabolic pathway essential for the maintenance of cellular and tissue homeostasis . AutophaThe mechanism of autophagosome biogenesis, specifically the origin and expansion of the autophagosomal membrane, has been a hot topic in the field . RecentlBif-1-depleted cells, suggesting the importance of Bif-1-mediated membrane budding for Atg9 trafficking. Interestingly, our previous data also show that the N-BAR domain of Bif-1 is, itself, not sufficient for nutrient starvation-induced Atg9 trafficking; therefore, protein complex formation through the SH3 domain of Bif-1 may be a critical step in regulating Atg9 vesicle formation during autophagy.The amino-terminal bin-amphiphysin-rvs (N-BAR) protein family, which forms a crescent-shape structure capable of deforming lipid bilayers, has emerged as a key regulator of membrane fission events . Bif-1  as a Bif-1-interacting protein and provide evidence that the Bif-1-DNM2-mediated membrane fission machinery plays a critical role in generating Atg9 vesicles during autophagy. We found that the Rab11-positive compartment serves as the reservoir for the Bif-1-DNM2-dependent generation of Atg9 vesicles required for proper autophagic flux.To understand the molecular mechanism of Atg9 vesicle generation, we performed tandem affinity purification using TAP-tagged -TEV-IgG-binding domain) Bif-1 fusion protein as bait. Mass spectrometry analysis identified several vesicle trafficking-related proteins as putative Bif-1-interacting proteins including a large GTPase, Dynamin (DNM) 2. Since DNMs are known to cooperate with N-BAR containing proteins during membrane fission , we deciDNM2 contains a proline-rich domain known to interact with SH3 domain-containing proteins . To deteDNM2 shRNAs effectively suppressed the expression of DNM2. Consistent with previous findings [Scr) cells  mouse fibroblasts with lentiviruses encoding Atg9a-EGFP. The resultant cells were treated with or without tamoxifen to obtain TKO/Atg9-GFP and control/Atg9-GFP cells. Depletion of DNMs was determined by immunoblotting  results in the accumulation of Atg9 in perinuclear Rab11-positive compartments. We have previously proposed that Bif-1 regulates Atg9-containing vesicle formation by mediating the fission of the Golgi membrane [It has been proposed that recycling endosome-like structures may serve as the mammalian Atg9 reservoirs to promote autophagosome formation . The impmembrane . Consistmembrane , 9 as weDNM2  under both nutrient-rich and nutrient-starved conditions in the absence of Dynasore. Notably, dynamin GTPase inhibitors including Dynasore have been shown to destabilize F-actin, disrupts lipid raft organization and inhibit membrane ruffling in a dynamin-independent manner [DNM or Atg9a depletion accumulates, rather than decreases, the levels of LC3-II. This result suggests that impaired LC3 lipidation in the presence of Dynasore may be an off-target effect of the inhibitor. Nonetheless, both Dynasore treatment as well as DNM or Atg9a depletion impair the degradation of autophagic substrate p62 under basal and starvation conditions to highlight the importance of this process for autophagic flux. Moreover, the observation that loss of DNMs or Atg9a increases the levels of LC3-II upon starvation in the presence of BafA1 suggests that Atg9 vesicles may promote a lysosome-independent LC3-II turnover (e.g. the delipidation of LC3-II on the growing phagophores) to complete autophagosome formation. Interestingly, it has been reported that Atg9 colocalizes with Atg2 and Atg18 at the growing edge of the phagophore, which have been implicated in autophagosome sealing [While our present data demonstrate the importance of the DNM-mediated membrane fission machinery for the regulation of Atg9 vesicles at the Rab11-positive reservoirs, DNM2 has also been proposed to regulate Atg9 vesicle generation at the plasma membrane . In thest manner , 29. Thet manner  could be sealing . Future DNM2, TRCN0000006650, 5′- ATGTAAGTGGTGACGATTCGC-3′; shDNM2 #2, TRC N0000006649, 5′-TTCGTGTTGATGTAGGACTGC-3′) were obtained from Dharmacon/Open Biosystems. Control scrambled shRNA (1864) [DNM triple knockout (TKO) murine fibroblasts [Atg9a–/– mouse embryonic fibroblasts [Antibodies were obtained from the following sources: mouse monoclonal anti-β-Actin , rabbit monoclonal anti-Atg9A , mouse monoclonal anti-Bif-1 , goat polyclonal anti-Dynamin II , rabbit polyclonal anti-Dynamin , mouse monoclonal anti-Flag , rabbit polyclonal anti-Flag , goat polyclonal anti-c-Myc , rabbit monoclonal anti-Myc-Tag , rabbit monoclonal anti-Rab5 , mouse monoclonal anti-Rab5 , rabbit monoclonal anti-Rab6 , rabbit monoclo- nal anti-Rab9 , rabbit monoclonal anti-Rab11 , rabbit polyclonal anti-TGN46 , mouse monoclonal anti-α-Tubulin , goat anti-mouse IgG conjugated with Alexa Fluor 405  or 568 , and goat anti-rabbit IgG conjugated with Alexa Fluor 405  or 568 . The Atg9A-GFP cDNA [A (1864)  was obtaA (1864) . Immortaroblasts  were genHeLa/S cells, 293T cells, mouse fibroblasts and mouse embryonic fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum and 1× Antibiotic Antimycotic Solution . Nucleofection was performed according to the manufacture's protocol. Lentivirus-mediated gene transduction and silencing were performed as previously described , 36. DNM3VO4, and protease inhibitors). Cell lysates were then subjected to purification and putative Bif-1-interacting proteins were identified by mass spectrometry as described previously [3COO)2, pH = 6.5; for Figure The human Bif-1 cDNA was subcloned into the EcoRI-Xho I site of pcDNA3.4-N-R1 TAP vector  and traneviously . For co-2 chamber. Fluorescent images were obtained using an OLYMPUS IX81 deconvolution microscope (63× oil-immersion lens) or a Leica AOBS SP8 laser-scanning confocal microscope (63× oil-immersion lens), deconvolved using SlideBook software (Intelligent Imaging Innovations) or Huygens deconvolution software (Scientific Volume Imaging) and analyzed using SlideBook software or and Imaris software (Bitplane). Electron microscopy was performed as described previously [For fluorescence and immunofluorescence microscopy, HeLa cells were grown overnight on Lab-TekII Chamber Slide . After each treatment, cells were fixed in paraformaldehyde-PBS, permeabilized with 50 μg/ml digitonin for 10 min, blocked with 10% normal goat serum for 1 h, incubated with the primary and the secondary antibodies and mounted with ProLong Gold Antifade Mountant ). For 3D time-lapse live cell imaging, cells were seeded on glass-bottom microwell dishes  after nucleofection, cultured for 24 h and pre-starved for 45 min followed by the addition of Dynasore (80 μM) to the medium. 3D images were taken at 30-sec intervals using a Leica AOBS SP8 laser-scanning confocal microscope with a heated COg at 4°C in order to obtain post-nuclear cell homogenates. The resultant post-nuclear cell homogenates (0.8 ml) were loaded on 6.4 ml of a 5–40% continuous OptiPrep  gradient and centrifuged at 36,000 rpm for 20 h at 4°C using a SW-41 swing rotor. Twelve fractions were collected from the top of the gradient and analyzed by immunoblotting with the indicated antibodies.Subcellular fractionation was performed as described previously . BrieflyTissue lysates were prepared in radio-immunoprecipitation assay buffer  containing protease and phosphatase inhibitors and subjected to immunoblotting The intensity of p62 and LC3-II were quantified by densitometry using Image Studio 5.0 software (LI-COR Biosciences) and normalized to β-actin. The percentages of (1) starvation-induced p62 decrease, (2) the lysosomal turnover of LC3-II under starvation and (3) the increase of LC3-II during starvation in the presence of BafA1 were calculated as follows: (1) (non-starved – starved)/non-starved × 100; (2) (starved with BafA1 – starved)/starved with BafA1 × 100; (3) (starved with BafA1 – non-starved with BafA1)/starved with BafA1 × 100.


Dexamethasone improves clinical outcomes in COVID-19 patients requiring supplementary oxygen. We investigated possible mechanisms of action by comparing sixteen plasma host response biomarkers in general ward patients before and after implementation of dexamethasone as standard of care. 48 patients without and 126 patients with dexamethasone treatment were sampled within 48 h of admission. Endothelial cell and coagulation activation biomarkers were comparable. Dexamethasone treatment was associated with lower plasma interleukin (IL)-6 and IL-1 receptor antagonist levels, whilst other inflammation parameters were not affected. These data argue against modification of vascular-procoagulant responses as an early mechanism of action of dexamethasone in COVID-19.The online version contains supplementary material available at 10.1186/s12931-022-02060-3. Dexamethasone decreases the risk of progression to invasive mechanical ventilation and mortality in hospitalized patients with coronavirus disease 2019 (COVID-19) requiring supplementary oxygen . IL interleukin, IL-1RA interleukin-1 receptor antagonist, MMP-8 matrix metalloproteinase-8, RAGE receptor for advanced glycation end-products, TREM-1 triggering receptor expressed on myeloid cells-1, VCAM-1 vascular cell adhesion molecule-1. * Reasons for missingness were discharged (41.3%), transfer to another hospital (5.8%), initiation of trial medication (10.8%) and no sampling (39.7%).