23 research outputs found
Oncogenic function of TBC1D15.
<p>(A) Tumor initiation titration. Defined numbers of TISCs stably expressing the indicated transgenes or lentivirus shRNAs were implanted subcutaneously into the dorsal hind flanks of NOG mice and tumor growth monitored for 60 days. Tumors greater than 25 mm<sup>3</sup> and which exhibited growth progression during the course of the study were scored as positive. (B) Tumor growth kinetics. TISCs (5× 10<sup>4</sup>) were implanted subcutaneously into NOG mice as in (A) and tumor volumes were measured on the indicated days. At least 6 tumors were measured for each cell line examined. (C) TBC1D15 expression in HCC patient tissue specimens. Tumors and matched, non-cancerous control tissues were processed by sectioning and immunostained using TBC1D15 antibody, then counterstained with hematoxylin to indicate cell nuclei. Below, high magnification image showing punctate localization of TBC1D15 (arrows) in noncancerous control tissue. Scale bar, 50 μm. (D) Stained tissues from patient specimens were scored for the degree of TBC1D15 immunopositivity. Brackets indicate statistically significant (**<i>P</i><0.01) comparisons between groups. The number of total cells scored across all samples (n = 17) is indicated for each group. (E) Box plot showing the expression of TBC1D15 in diverse tumor types and matched normal tissues. Red line indicates the median, box edges the 25–75 percentiles. Whiskers represent 1.5×interquartile range (IQR) above the third quartile or below the first quartile. Outliers (blue circles or black triangles) are shown when outside this range. (F) Scatter plots showing results from meta-analysis comparing the expression levels of <i>TBC1D15</i> and <i>NANOG</i> across multiple tumor samples and in matched, non-tumor control tissues.</p
Starvation- and p53-induced degradation of TBC1D15.
<p>(A) Flag- TBC1D15 was expressed alone or with myc-p53 as indicated, followed by cell lysis and analysis by immunoblotting. (B) p53 transactivation function mediates destabilization of myc-TBC1D15. HEK-293A cells were transfected with myc-TBC1D15 and either empty vector or Flag-p53 (human), wild-type murine p53 or the transactivation-deficient point mutant p53D278N, followed by lysis and immunoblotting. (C) Cells were transfected with Flag-TBC1D15 and either empty vector or myc-p53, followed by exposure for 6 hr to autophagy inhibitor Bafilomycin A1 (100 nM) or proteasome inhibitor lactacystin (5 μM). (D) HEK-293A cells expressing myc-TBC1D15 were exposed for 6 hr to DMSO vehicle or to the mTOR inhibitors rapamycin (100 nM) or PP242 (1 μM), then lysed and analyzed by immunoblotting. (E) Cells were transfected with myc-TBC1D15 and cultured in complete media or shifted to glucose- and amino acid-free media for 6 hr in the absence or presence of Bafilomycin A1, as indicated. (F) Elevated levels of TBC1D15 in <i>Atg5</i>- and <i>p53</i>-deficient livers. Livers were surgically resected from p53-deficient mice or from mice with hepatocyte-specific deletion of <i>Atg5</i> (<i>Atg5<sup>fl</sup></i><sup>/fl</sup>; <i>Alb-cre</i>) or littermate controls. Liver lysates were resolved by SDS-PAGE and analyzed by immunoblotting.</p
Conceptual model of TBC1D15 function in TISC-mediated oncogenesis.
<p>The transition from an untransformed hepatocyte to a TISC is accompanied by an anabolic shift and increased levels of the TBC1D15 oncoprotein, which destabilizes the Numb-p53 complex to promote deregulated self-renewal and oncogenesis.</p
The TBC1D15 Oncoprotein Controls Stem Cell Self-Renewal through Destabilization of the Numb-p53 Complex
<div><p>Stem cell populations are maintained through self-renewing divisions in which one daughter cell commits to a specific fate while the other retains the multipotent characteristics of its parent. The p53 tumor suppressor, in conjunction with its interacting partner protein Numb, preserves this asymmetry and functions as a vital barrier against the unchecked expansion of tumor stem cell pools; however, little is known about the biological control of the Numb-p53 interaction. We show here that Numb and p53 are the constituents of a high molecular mass complex, which is disintegrated upon activation of aPKCζ, a Numb kinase. Using large-scale affinity purification and tandem mass spectrometry, we identify TBC1D15 as a Numb-associated protein and demonstrate that its amino-terminal domain disengages p53 from Numb, triggering p53 proteolysis and promoting self-renewal and pluripotency. Cellular levels of TBC1D15 are diminished upon acute nutrient deprivation through autophagy-mediated degradation, indicating that TBC1D15 serves as a conduit through which cellular metabolic status is linked to self-renewal. The profound deregulation of TBC1D15 expression exhibited in a diverse array of patient tumors underscores its proposed function as an oncoprotein.</p> </div
The Numb-binding domain of TBC1D15 targets p53 for proteolysis.
<p>(A) Following transfection with the indicated expression vectors, HEK-293A cells were exposed to vehicle or Nutlin-3 (10 μM) for 16 hr as indicated, then lysed and analyzed by immunoblotting. (B) Depletion of endogenous <i>TBC1D15</i> increases steady-state levels of p53. HEK-293A cells were stably transduced with lentivirus vectors encoding a non-targeting, scrambled shRNA control (sh-<i>Scr</i>) or two different shRNAs targeting <i>TBC1D15</i>, or transfected with Flag-p53 or myc-TBC1D15, followed by preparation of lysates, SDS-PAGE and immunoblotting using the indicated antibodies. (C) Depletion of TBC1D15 sensitizes cells to apoptosis. HEK-293A cells harboring a non-targeting shRNA or sh1-<i>TBC1D15,</i> or expressing either myc-TBC1D15 or Flag-p53, were treated with DMSO vehicle or with etoposide (34 μM, 16 hr) followed by propidium iodide staining and FACS analysis. The percentage of sub-G1 DNA content is indicated in each histogram. (D) The amino-terminal domain of TBC1D15 targets p53 for proteolysis. Myc-p53 was expressed in HEK-293A cells with empty vector or with the indicated Flag-TBC1D15 constructs. Lysates were immunoprecipitated using either anti-Numb or anti-myc agarose. GFP was co-transfected as an internal control for transfection efficiency. (E) Functional dissection of the TBC1D15 amino-terminal domain in p53 destabilization. Myc-p53 was transfected with GFP or with the indicated GFP-TBC1D15 fusions, followed by lysis, Numb immunoprecipitation and immunoblotting. Lysates represent 10% of the input volume used in the immunoprecipitation.</p
Hepatitis C Virus Translation Preferentially Depends on Active RNA Replication
<div><p>Hepatitis C virus (HCV) RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER) in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome.</p> </div
Double-labeling of newly-synthesized HCV RNA and newly synthesized viral peptides in JFH1-infected Huh-7.5 cells.
<p>Huh7.5 cells were infected with HCV JFH-1 strain for 2 days, and then were labeled with Cy5-UTP and BODIPY-FL-Lys-tRNA in the presence of actinomycin D and hippuristanol, which inhibit host RNA and protein synthesis, respectively. The cells were kept in 37°C chamber supplied with CO<sub>2</sub> for live cell imaging on a Zeiss LSM 510 laser scanning confocal microscope. Images were taken after 10–40 minutes of chase. Newly-synthesized HCV RNA was the first to be detected (as shown in red) and was in a perinuclear pattern. Newly-translated HCV viral peptides (as shown in green) were detected at later time points, completely co-localized with the sites of RNA synthesis. No significant amount of Cy5-UTP and BODIPY-FL-Lys-tRNA labeling could be detected in naïve Huh7.5 cells (as a negative control) in the presence of actinomycin D and hippuristanol.</p
HCV RNA translation is dependent on the RNA transcription.
<p>(A), Mock- or Benzothiadiazine-treated Huh-N1b and Huh-Neo cells were labeled by <sup>35</sup>S-Methionine for 4 hours, and followed by immunoprecipitation with anti-NPT or anti-NS3 antibodies or sera from hepatitis C patients. The immunoprecipitates were separated by SDS-PAGE and detected by autoradiography. (B) The intracellular replicon RNA was detected by Northern blotting. (C) Huh7 cells transfected with <i>in vitro</i> transcribed Rep or the replication-defective Rep*GND RNA were metabolically labeled with <sup>35</sup>S-Methionine for 14 hours and followed by immunoprecipitation with anti-NS3 antibody. (D) The amounts of the intracellular HCV RNA in panel (C) were determined by realtime RT-PCR and Nothern blotting. The relative ratios of the HCV RNA/GAPDH mRNA in the different cells are presented. E) Structures of the bi-cistronic replicon reporter constructs used. Time course studies of the luciferase activity in cells transfected with constructs 1 and 2 (panel (F)) and constructs 3 and 4 (panel (G)) were measured by dual luciferase assay at various time points after transfection. The ratios of the FFluc/Rluc (F) or Rluc/FFluc (G) are presented. Error bars represent +/− standard deviation.</p
The translocation of newly-synthesized HCV RNA.
<p>HCV replicon cells were labeled with BrUTP (A) or <sup>3</sup>H-Uridine (B) in the presence of actinomycin D and chased for up to 180 minutes. (A) Immunofluorescence staining with anti-BrdU and other organelle antibodies shows the colocalization of BrU-labeled HCV RNA with ER initially (30 min) and then with Golgi (180 min). (B) Fractionation of ER and Golgi by sucrose gradient. Fraction numbers and their gradient positions are noted at the bottom. <sup>3</sup>H-Uridine-labeled RNA in the ER (fraction 4) and the Golgi (fraction 6–8) fractions were collected, and the radioactivity of <sup>3</sup>H-Uridine-labeled RNA was counted. Immunoblotting of ER and Golgi makers demonstrates the separation of ER and Golgi by sucrose gradient fractionation.</p
The proposed model of coupled replication/translation of HCV RNA.
<p>A proposed model of HCV replication-translation complex “replicasome”. As reported, HCV replication complexes are assembled at ER, and then bud into the ER lumen. Consequently, (A) HCV RNA replication is first initiated in the multi-layered vesicle structure derived from the ER membrane. (B) The newly synthesized HCV RNA is translated around the ER-derived vesicle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043600#pone.0043600-Sir1" target="_blank">[39]</a>, where there are membrane-associated ribosomes. Benzothiadiazine blocks HCV RNA transcription and therefore decreases translation. (C) The newly-synthesized HCV RNA is later transported away from ER; nocodazole inhibits this transportation. HCV RNA is then transported to Golgi-derived membrane and/or then the lipid droplet (LD) for packaging and assembly of virus particles.</p