PURPOSE Androgen receptor splice variant 7 (AR-V7) results in a truncated receptor, which leads to ligand-independent constitutive activation that is not inhibited by anti-androgen therapies, including abiraterone or enzalutamide. Given that previous reports suggested that circulating tumor cell (CTC) AR-V7 detection is a poor prognostic indicator for the clinical efficacy of secondary hormone therapies, we conducted a prospective multicenter validation study. PATIENTS AND METHODS PROPHECY ( ClinicalTrials.gov identifier: NCT02269982) is a multicenter, prospective-blinded study of men with high-risk mCRPC starting abiraterone acetate or enzalutamide treatment. The primary objective was to validate the prognostic significance of baseline CTC AR-V7 on the basis of radiographic or clinical progression free-survival (PFS) by using the Johns Hopkins University modified-AdnaTest CTC AR-V7 mRNA assay and the Epic Sciences CTC nuclear-specific AR-V7 protein assay. Overall survival (OS) and prostate-specific antigen responses were secondary end points. RESULTS We enrolled 118 men with mCRPC who were starting abiraterone or enzalutamide treatment. AR-V7 detection by both the Johns Hopkins and Epic AR-V7 assays was independently associated with shorter PFS (hazard ratio, 1.9 [95% CI, 1.1 to 3.3; P = .032] and 2.4 [95% CI, 1.1 to 5.1; P = .020], respectively) and OS (hazard ratio, 4.2 [95% CI, 2.1 to 8.5] and 3.5 [95% CI, 1.6 to 8.1], respectively) after adjusting for CTC number and clinical prognostic factors. Men with AR-V7–positive mCRPC had fewer confirmed prostate-specific antigen responses (0% to 11%) or soft tissue responses (0% to 6%). The observed percentage agreement between the two AR-V7 assays was 82%. CONCLUSION Detection of AR-V7 in CTCs by two blood-based assays is independently associated with shorter PFS and OS with abiraterone or enzalutamide, and such men with mCRPC should be offered alternative treatments.
The integrin receptor ␣v5 controls two independent forms of interactions of the retinal pigment epithelium (RPE) with adjacent photoreceptor outer segments that are essential for vision. ␣v5 localizes specifically to apical microvilli of the RPE and contributes to retinal adhesion that maintains RPE contacts with intact outer segments at all times. Additionally, ␣v5 synchronizes diurnal bursts of RPE phagocytosis that clear photoreceptor outer segment fragments (POS) shed in a circadian rhythm. Dependence of retinal phagocytosis and adhesion on ␣v5 receptors suggests that the extracellular matrix ensheathing RPE microvilli contains ligands for this integrin. Here we studied mice lacking expression of functional MFG-E8 to test the contribution of this integrin ligand to ␣v5 functions in the retina. Lack of MFG-E8 only minimally reduced retinal adhesion. In contrast, lack of MFG-E8, like lack of ␣v5 receptor, eliminated ␣v5 downstream signaling involving the engulfment receptor MerTK and peak POS phagocytosis, both of which follow light onset in wild-type retina. MFG-E8-deficient RPE in primary culture retained normal epithelial morphology and levels of apical ␣v5 receptors, but showed impaired binding and engulfment of isolated POS. Soluble or POS-bound recombinant MFG-E8 was sufficient to fully restore phagocytosis by MFG-E8-deficient RPE. Furthermore, MFG-E8 supplementation strongly increased POS binding by wild-type and MerTK-deficient RPE, but did not affect POS binding by RPE lacking ␣v5. Thus, MFG-E8 stimulates rhythmic POS phagocytosis by ligating apical ␣v5 receptors of the RPE. These results identify MFG-E8 as the first extracellular ligand in the retina that is essential for diurnal POS phagocytosis.adhesion ͉ photoreceptors ͉ retinal pigment epithelium ͉ circadian rhythm ͉ outer segment T he phototransduction machinery of retinal rod and cone photoreceptor neurons localizes to their outer segment portions, which face the apical surface of the retinal pigment epithelium (RPE). Interactions of RPE cells with outer segments support photoreceptor function. RPE cells employ apical surface receptors at all times to promote retinal adhesion stabilizing alignment and RPE microvilli interdigitation with outer segments. Once a day, RPE cells use their apical phagocytic receptors and engulfment machinery to respond to circadian shedding of photoreceptor outer segment fragments (POS) with a vigorous burst of POS phagocytosis. Disruption of retinal adhesion in persistent retinal detachment or incomplete POS clearance by the RPE cause outer segment degeneration and photoreceptor apoptosis (1). Thus, intact receptor-mediated interactions of RPE cells with POS are critical to maintaining vision for life.␣v5 integrin is the only integrin family receptor at the apical surface of the RPE in rodent and human retina (2, 3). 5 integrin knockout (5 Ϫ/Ϫ ) mice show greatly weakened retinal adhesion at all times of day (4). Furthermore, 5 Ϫ/Ϫ retina lacks the daily rhythm of RPE phagocytosis because 5 Ϫ/Ϫ RPE fa...
Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of the aminoacyl-tRNA-eEF1A-GTP ternary complex to the ribosomal A-site and has been suggested to facilitate the clearance of deacyl-tRNA from the E-site. Here we present the crystal structure of Saccharomyces cerevisiae eEF3, showing that it consists of an amino-terminal HEAT repeat domain, followed by a four-helix bundle and two ABC-type ATPase domains, with a chromodomain inserted in ABC2. Moreover, we present the cryo-electron microscopy structure of the ATP-bound form of eEF3 in complex with the post-translocational-state 80S ribosome from yeast. eEF3 uses an entirely new factor binding site near the ribosomal E-site, with the chromodomain likely to stabilize the ribosomal L1 stalk in an open conformation, thus allowing tRNA release.Protein synthesis requires, in general, only two canonical GTPase elongation factors. eEF1A (known as EF-Tu in prokaryotes) recruits cognate aminoacyl-tRNAs to the A-site of the ribosome, and, after peptidyl transfer, eEF2 (EF-G in prokaryotes) catalyses translocation of the messenger RNA and the transfer RNAs from the A-and P-sites to the P-and E-sites. In contrast to the canonical factors, eEF3 has an ATPase activity that is stimulated by ribosomes. It interacts with both ribosomal subunits 1-3 , competes with eEF2 for binding to ribosomes, and stimulates eEF1A-dependent binding of cognate aminoacyltRNA to the ribosomal A-site 1,4 . Because, according to the allosteric three-site model of the ribosomal elongation cycle, E-site release is required for efficient A-site binding, it has been suggested that eEF3 functions as a so-called 'E-site factor' 4,5 . Moreover, ATP hydrolysis by eEF3 is required in every elongation cycle to allow chasing of deacyltRNA from the E-site 4 .eEF3 belongs to the family of ABC (ATP-binding cassette) proteins that includes proteins involved in transport across membranes, DNA repair, and translation. The membrane proteins of this class especially represent important targets for development of novel therapeutic strategies. The proteins contain ATP/ADP-binding ABC domains, which convert chemical energy derived from binding of ATP or its hydrolysis into a 'powerstroke' of mechanical energy 6 . ABC proteins function as either homodimers or as twin-cassette proteins with two ABC domains within the same polypeptide.The ribosome exhibits very dynamic behaviour, such as the ratchet movement 7 or the movement of the L1 and the L7/L12 stalks [8][9][10][11] . Hence, an intriguing question is how the interaction of eEF3 with the ribosome is correlated with its dynamic properties as an ABC protein, and how the energy derived from binding/hydrolysis of ATP is used for its function. Crystal structure of eEF3Three crystal structures of residues 1-980 of eEF3 in the apo state (2.7 Å ), in complex with ADP (2.4 Å ), or in complex with the nonhydrolysable ATP analogue ADPNP (3...
Rad23 and Rpn10 play synergistic roles in the recognition of ubiquitinated proteins by the proteasome, and loss of both proteins causes growth and proteolytic defects. However, the physiological targets of Rad23 and Rpn10 have not been well defined. We report that rad23⌬ rpn10⌬ is unable to grow in the presence of translation inhibitors, and this sensitivity was suppressed by translation elongation factor 1A (eEF1A). This discovery suggested that Rad23 and Rpn10 perform a role in translation quality control. Certain inhibitors increase translation errors during protein synthesis and cause the release of truncated polypeptide chains. This effect can also be mimicked by ATP depletion. We determined that eEF1A interacted with ubiquitinated proteins and the proteasome following ATP depletion. eEF1A interacted with the proteasome subunit Rpt1, and the turnover of nascent damaged proteins was deficient in rpt1. An eEF1A mutant (eEF1A D156N ) that conferred hyperresistance to translation inhibitors was much more effective at eliminating damaged proteins and was detected in proteasomes in untreated cells. We propose that eEF1A is well suited to detect and promote degradation of damaged proteins because of its central role in translation elongation. Our findings provide a mechanistic foundation for defining how cellular proteins are degraded cotranslationally.Rad23 and Rpn10 can interact with multiubiquitinated proteins (6, 28, 38) and the proteasome (32), and several recent studies have indicated that they contribute to the degradation of ubiquitinated proteins by the proteasome (6, 9, 21, 24). Loss of both proteins results in temperature-sensitive growth, defects in proteolysis, and a delay in the G 2 phase of the cell cycle (22). We isolated yeast TEF1, a gene encoding the eukaryotic translation elongation factor 1A (eEF1A) (15), as a dosage suppressor of the cold (13°C) sensitivity of rad23⌬ rpn10⌬ (22). eEF1A promotes translation elongation through the binding and release of aminoacyl tRNAs, in a process that is coupled to GTP hydrolysis (15). In addition to its well-characterized role in translation elongation, in vitro studies showed that eEF1A could bind nascent as well as unfolded peptides and proteins (17,23). eEF1A might possess a chaperone-like activity that prevents the aggregation of nascent polypeptide chains (4), since it could bind an unfolded protein but not a correctly folded counterpart (17). eEF1A could also stimulate the degradation of N␣-acetylated proteins (12). The isolation of eEF1A as a suppressor of rad23⌬ rpn10⌬ suggested that it performs a central role in monitoring the accuracy of protein synthesis. These studies also revealed an important function for Rad23 and Rpn10 in protein synthesis quality control.A significant fraction of newly synthesized proteins is degraded cotranslationally (29,33,35). These nascent damaged proteins can be ubiquitinated while bound to the ribosome (31), demonstrating that there exists a close coupling between the pathways of protein synthesis and protein degrada...
Accumulation of indigestible lipofuscin and decreased mitochondrial energy production are characteristic age-related changes of post-mitotic retinal pigment epithelial (RPE) cells in the human eye. To test whether these two forms of age-related impairment have interdependent effects, we quantified the ATPdependent phagocytic function of RPE cells loaded or not with the lipofuscin component A2E and inhibiting or not mitochondrial ATP synthesis either pharmacologically or genetically. We found that physiological levels of lysosomal A2E reduced mitochondrial membrane potential and inhibited oxidative phosphorylation (OXPHOS) of RPE cells. Furthermore, in media with physiological concentrations of glucose or pyruvate, A2E significantly inhibited phagocytosis. Antioxidants reversed these effects of A2E, suggesting that A2E damage is mediated by oxidative processes. Because mitochondrial mutations accumulate with aging, we generated novel genetic cellular models of RPE carrying mitochondrial DNA point mutations causing either moderate or severe mitochondrial dysfunction. Exploring these mutant RPE cells we found that, by itself, only the severe but not the moderate OXPHOS defect reduces phagocytosis. However, sub-toxic levels of lysosomal A2E are sufficient to reduce phagocytic activity of RPE with moderate OXPHOS defect and cause cell death of RPE with severe OXPHOS defect. Taken together, RPE cells rely on OXPHOS for phagocytosis when the carbon energy source is limited. Our results demonstrate that A2E accumulation exacerbates the effects of moderate mitochondrial dysfunction. They suggest that synergy of sub-toxic lysosomal and mitochondrial changes in RPE cells with age may cause RPE dysfunction that is known to contribute to human retinal diseases like agerelated macular degeneration. Retinal pigment epithelial (RPE)5 cells form a polarized monolayer epithelium between the photoreceptors of the neurosensory retina and the choroidal capillary bed. Daily phagocytosis of outer segment (OS) tips shed by adjacent photoreceptors is a vital task of the RPE (recently reviewed by Strauss (1)). RPE cells are post-mitotic and face each ϳ30 photoreceptor outer segments in the human eye, all of which shed their distal tip containing stacked membrane disks once a day. Diurnal phagocytosis and digestion of thousands of OS disks for life renders RPE cells the most active phagocytes in the body. Photoreceptor function strictly depends on efficient RPE phagocytosis of spent OS. Complete failure of RPE cells to engulf OS causes rapid photoreceptor degeneration in the Royal College of Surgeons rat (2-4). Impaired RPE phagocytosis also contributes to human retinal disease such as retinitis pigmentosa and, likely, age-related macular degeneration (5, 6).The continuous nature of outer segment renewal implies that any delay in OS removal by aged or damaged RPE will gradually cause OS components to accumulate. RPE cells are at risk for oxidative damage due to their location in the highly oxygenated environment of the outer retina and th...
The Lec35 gene product (Lec35p) is required for utilization of the mannose donor mannose-P-dolichol (MPD) in synthesis of both lipid-linked oligosaccharides (LLOs) and glycosylphosphatidylinositols, which are important for functions such as protein folding and membrane anchoring, respectively. The hamster Lec35 gene is shown to encode the previously identified cDNA SL15, which corrects the Lec35 mutant phenotype and predicts a novel endoplasmic reticulum membrane protein. The mutant hamster alleles Lec35.1 and Lec35.2 are characterized, and the human Lec35 gene (mannose-P-dolichol utilization defect 1) was mapped to 17p12-13. To determine whether Lec35p was required only for MPD-dependent mannosylation of LLO and glycosylphosphatidylinositol intermediates, two additional lipid-mediated reactions were investigated: MPD-dependent C-mannosylation of tryptophanyl residues, and glucose-P-dolichol (GPD)-dependent glucosylation of LLO. Both were found to require Lec35p. In addition, the SL15-encoded protein was selective for MPD compared with GPD, suggesting that an additional GPD-selective Lec35 gene product remains to be identified. The predicted amino acid sequence of Lec35p does not suggest an obvious function or mechanism. By testing the water-soluble MPD analog mannose--1-P-citronellol in an in vitro system in which the MPD utilization defect was preserved by permeabilization with streptolysin-O, it was determined that Lec35p is not directly required for the enzymatic transfer of mannose from the donor to the acceptor substrate. These results show that Lec35p has an essential role for all known classes of monosaccharide-P-dolichol-dependent reactions in mammals. The in vitro data suggest that Lec35p controls an aspect of MPD orientation in the endoplasmic reticulum membrane that is crucial for its activity as a donor substrate.
Cancer stem cells, capable of self-renewal and multipotent differentiation, influence tumor behavior through a complex balance of symmetric and asymmetric cell divisions. Mechanisms regulating the dynamics of stem cells and their progeny in human cancer are poorly understood. In Drosophila, mutation of brain tumor (brat) leads to loss of normal asymmetric cell division by developing neural cells and results in a massively enlarged brain composed of neuroblasts with neoplastic properties. Brat promotes asymmetric cell division and directs neural differentiation at least partially through its suppression on Myc. We identified TRIM3 (11p15.5) as a human ortholog of Drosophila brat and demonstrate its regulation of asymmetric cell division and stem cell properties of glioblastoma (GBM), a highly malignant human brain tumor. TRIM3 gene expression is markedly reduced in human GBM samples, neurosphere cultures and cell lines and its reconstitution impairs growth properties in vitro and in vivo. TRIM3 expression attenuates stem-like qualities of primary GBM cultures, including neurosphere formation and the expression of stem cell markers CD133, Nestin and Nanog. In GBM stem cells, TRIM3 expression leads to a greater percentage dividing asymmetrically rather than symmetrically. As with Brat in Drosophila, TRIM3 suppresses c-Myc expression and activity in human glioma cell lines. We also demonstrate a strong regulation of Musashi-Notch signaling by TRIM3 in GBM neurospheres and neural stem cells that may better explain its effect on stem cell dynamics. We conclude that TRIM3 acts as a tumor suppressor in GBM by restoring asymmetric cell division.
The translation elongation machinery in fungi differs from other eukaryotes in its dependence upon eukaryotic elongation factor 3 (eEF3). eEF3 is essential in vivo and required for each cycle of the translation elongation process in vitro. Models predict eEF3 affects the delivery of cognate aminoacyl-tRNA, a function performed by eEF1A, by removing deacylated tRNA from the ribosomal Exit site. To dissect eEF3 function and its link to the A-site activities of eEF1A, we have identified a temperature-sensitive allele of the YEF3 gene. The F650S substitution, located between the two ATP binding cassettes, reduces both ribosome-dependent and intrinsic ATPase activities. In vivo this mutation increases sensitivity to aminoglycosidic drugs, causes a 50% reduction of total protein synthesis at permissive temperatures, slows run-off of polyribosomes, and reduces binding to eEF1A. Reciprocally, excess eEF3 confers synthetic slow growth, increased drug sensitivity, and reduced translation in an allele specific fashion with an E122K mutation in the GTP binding domain of eEF1A. In addition, this mutant form of eEF1A shows reduced binding of eEF3. Thus, optimal in vivo interactions between eEF3 and eEF1A are critical for protein synthesis.
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