Galectin-3 and prohibitin 1 are autoantigens in IgG4-related cholangitis without clear-cut protective effects against toxic bile acids

Background and aimsIgG4-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4-related disease, a systemic B cell-driven fibro-inflammatory disorder. Four autoantigens have recently been described in IgG4-RD: annexin A11, galectin-3, laminin 511-E8, and prohibitin 1. We have previously reported a protective role of annexin A11 and laminin 511-E8 in human cholangiocytes against toxic bile acids. Here, we explored the potentially protective role of the carbohydrate-binding lectin galectin-3 and the scaffold proteins prohibitins 1 and 2.MethodsAnti-galectin-3, anti-prohibitin 1 and 2 autoantibody positivity in IRC and healthy and disease (primary sclerosing cholangitis (PSC)) control sera was assessed by ELISA/liquid chromatography–tandem mass spectrometry (LC-MS/MS). Human H69 cholangiocytes were subjected to short hairpin RNA (shRNA) knockdown targeting galectin-3 (LGALS3), prohibitin 1 (PHB1), and prohibitin 2 (PHB2). H69 cholangiocytes were also exposed to recombinant galectin-3, the inhibitor GB1107, recombinant prohibitin 1, and the pan-prohibitin inhibitor rocaglamide. Protection against bile acid toxicity was assessed by intracellular pH (pHi) measurements using BCECF-AM, 22,23-3H-glycochenodeoxycholic acid (3H-GCDC) influx, and GCDC-induced apoptosis using Caspase-3/7 assays.ResultsAnti-galectin-3 autoantibodies were detected in 13.5% of individuals with IRC but not in PSC. Knockdown of LGALS3 and galectin-3 inhibition with GB1107 did not affect pHi, whereas recombinant galectin-3 incubation lowered pHi. LGALS3 knockdown increased GCDC-influx but not GCDC-induced apoptosis. GB1107 reduced GCDC-influx and GCDC-induced apoptosis. Recombinant galectin-3 tended to decrease GCDC-influx and GCDC-induced apoptosis. Anti-prohibitin 1 autoantibodies were detected in 61.5% and 35.7% of individuals with IRC and PSC, respectively. Knockdown of PHB1, combined PHB1/2 KD, treatment with rocaglamide, and recombinant prohibitin 1 all lowered pHi. Knockdown of PHB1, PHB2, or combined PHB1/2 did not alter GCDC-influx, yet knockdown of PHB1 increased GCDC-induced apoptosis. Conversely, rocaglamide reduced GCDC-influx but did not attenuate GCDC-induced apoptosis. Recombinant prohibitin 1 did not affect GCDC-influx or GCDC-induced apoptosis. Finally, anti-galectin-3 and anti-prohibitin 1 autoantibody pretreatment did not lead to increased GCDC-influx.ConclusionsA subset of individuals with IRC have autoantibodies against galectin-3 and prohibitin 1. Gene-specific knockdown, pharmacological inhibition, and recombinant protein substitution did not clearly disclose a protective role of these autoantigens in human cholangiocytes against toxic bile acids. The involvement of these autoantibodies in processes surpassing epithelial secretion remains to be elucidated

Chk1 and 14-3-3 proteins inhibit atypical E2Fs to prevent a permanent cell cycle arrest

The atypical E2Fs, E2F7 and E2F8, act as potent transcriptional repressors of DNA replication genes providing them with the ability to induce a permanent S-phase arrest and suppress tumorigenesis. Surprisingly in human cancer, transcript levels of atypical E2Fs are frequently elevated in proliferating cancer cells, suggesting that the tumor suppressor functions of atypical E2Fs might be inhibited through unknown post-translational mechanisms. Here, we show that atypical E2Fs can be directly phosphorylated by checkpoint kinase 1 (Chk1) to prevent a permanent cell cycle arrest. We found that 14-3-3 protein isoforms interact with both E2Fs in a Chk1-dependent manner. Strikingly, Chk1 phosphorylation and 14-3-3-binding did not relocate or degrade atypical E2Fs, but instead, 14-3-3 is recruited to E2F7/8 target gene promoters to possibly interfere with transcription. We observed that high levels of 14-3-3 strongly correlate with upregulated transcription of atypical E2F target genes in human cancer. Thus, we reveal that Chk1 and 14-3-3 proteins cooperate to inactivate the transcriptional repressor functions of atypical E2Fs. This mechanism might be of particular importance to cancer cells, since they are exposed frequently to DNA-damaging therapeutic reagents

Chaperonin CCT Checkpoint Function in Basal Transcription Factor TFIID Assembly

International audienc

Saccharomyces cerevisiae Sof1p Associates with 35S Pre-rRNA Independent from U3 snoRNA and Rrp5p

Sof1p is a trans-acting protein that is essential for biogenesis of the 40S ribosomal subunits in the yeast Saccharomyces cerevisiae. Because of its involvement in the early cleavage steps of precursor rRNA, its interaction with Nop1p and its ability to coprecipitate U3 snoRNA, Sof1p has so far been regarded as a protein that is specific to the U3 snoRNP. To determine whether a site exists within U3 snoRNA with which Sof1p directly or indirectly associates, we studied the ability of ProtA-tagged Sof1p to coimmunoprecipitate mutant versions of U3 snoRNA. None of the tested mutations had a significant effect on the recovery of mutant U3 from cell extracts. Further coimmunoprecipitation experiments, using cells that could be genetically depleted for either Sof1p or U3 snoRNA demonstrated that the two factors associate independently of each other with the 35S precursor RNA. Indeed, association between Sof1p and U3 snoRNA was abolished in cells in which 35S pre-rRNA transcription was blocked. Finally, we found that an overall reduction in the levels of box C/D snoRNPs by genetic depletion of the common Nop58p protein did not affect coprecipitation of 35S pre-rRNA by Sof1p. From these data, we conclude that Sof1p does not assemble into the 90S preribosome as part of the U3, or any other box C/D, snoRNP. The early and independently assembling trans-acting factor Rrp5p also proved to be dispensable for assembly of Sof1p

U3 snoRNP and Rrp5p associate independently with Saccharomyces cerevisiae 35S pre-rRNA, but Rrp5p is essential for association of Rok1p

Biogenesis of eukaryotic ribosomal subunits proceeds via a series of precursor ribonucleoprotein particles that correspond to different stages in the maturation pathway. The different pre-ribosomal particles each contain a distinct complement of non-ribosomal, trans-acting factors that are crucial for correct and efficient progress of the maturation process. Although in recent years we have gained considerable insight into the composition of the pre-ribosomal particles, our knowledge how the ordered association with and their dissociation from the pre-ribosome of these trans-acting factors is controlled is still quite limited. Here, we have studied the mutual dependence between three of these factors, Rrp5p, U3 snoRNP and Rok1p, all essential for the early stages of pre-rRNA processing/assembly, for association with the 35S pre-rRNA in Saccharomyces cerevisiae. Using co-immunoprecipitation assays, we show that Rrp5p and U3 snoRNP associate independently of each other and that the two factors do not detectably interact prior to incorporation into the pre-ribosome. In contrast, association of the putative RNA helicase Rok1p, which is known to genetically interact with Rrp5p, is absolutely dependent on the presence of the latter protein but does not require U3

Proteomic profiling of two distinct populations of extracellular vesicles isolated from human seminal plasma

Body fluids contain many populations of extracellular vesicles (EV) that differ in size, cellular origin, molecular composition, and biological activities. EV in seminal plasma are in majority originating from prostate epithelial cells, and hence are also referred to as prostasomes. Nevertheless, EV are also contributed by other accessory sex glands, as well as by the testis and epididymis. In a previous study, we isolated EV from seminal plasma of vasectomized men, thereby excluding contributions from the testis and epididymis, and identified two distinct EV populations with diameters of 50 and 100 nm, respectively. In the current study, we comprehensively analyzed the protein composition of these two EV populations using quantitative Liquid Chromatography-Mass Spectrometry (LC-MS/MS). In total 1558 proteins were identified. Of these, ≈45% was found only in the isolated 100 nm EV, 1% only in the isolated 50 nm EV, and 54% in both 100 nm and 50 nm EV. Gene ontology (GO) enrichment analysis suggest that both originate from the prostate, but with distinct biogenesis pathways. Finally, nine proteins, including KLK3, KLK2, MSMB, NEFH, PSCA, PABPC1, TGM4, ALOX15B, and ANO7, with known prostate specific expression and alternate expression levels in prostate cancer tissue were identified. These data have potential for the discovery of EV associated prostate cancer biomarkers in blood

Rrp5p, a trans-acting factor in yeast ribosome biogenesis, is an RNA-binding protein with a pronounced preference for U-rich sequences

Rrp5p is a trans-acting factor important for biogenesis of both the 40S and 60S subunit of the Saccharomyces cerevisiae ribosome. The protein contains 12 tandemly repeated S1 RNA binding motifs in its N-terminal region, suggesting the ability to interact directly with the pre-rRNA. In vitro binding studies, using immunopurified Rrp5p and in vitro transcribed, (32)P-UTP-labeled RNA fragments, revealed that Rrp5p is a general RNA-binding protein with a strong preference for single-stranded sequences rich in uridines. Co-immunoprecipitation studies in yeast cells expressing ProtA-tagged Rrp5p showed that the protein is still associated with pre-ribosomal particles containing 27SA2 pre-rRNA but not with particles containing the 27SB precursor. Thus, Rrp5p appears to dissociate from the 66S pre-ribosome upon or immediately after further processing of 27SA2 pre-rRNA, suggesting the presence of (an) important binding site(s) within the 3′-terminal portion of ITS1. The location of these possible binding site(s) was further delimited using rrp2-1 mutant cells, which accumulate the 5′-extended 5.8S pre-rRNA species. The results indicate that association of Rrp5p with the pre-ribosome is abolished upon removal of a 30-nt region downstream from site A2, which contains two short, single-stranded U stretches. Sequence comparison shows that only the most 5′ of these two U-rich stretches is conserved among yeast species whose ITS1 can functionally replace the S. cerevisiae spacer. The implications for the role of Rrp5p in yeast ribosome biogenesis are discussed

The RNA catabolic enzymes Rex4p, Rnt1p, and Dbr1p show genetic interaction with trans-acting factors involved in processing of ITS1 in Saccharomyces cerevisiae pre-rRNA

Eukaryotes have two types of ribosomes containing either 5.8S(L) or 5.8S(S) rRNA that are produced by alternative pre-rRNA processing. The exact processing pathway for the minor 5.8S(L) rRNA species is poorly documented. We have previously shown that the trans-acting factor Rrp5p and the RNA exonuclease Rex4p genetically interact to influence the ratio between the two forms of 5.8S rRNA in the yeast Saccharomyces cerevisiae. Here we report a further analysis of ITS1 processing in various yeast mutants that reveals genetic interactions between, on the one hand, Rrp5p and RNase MRP, the endonuclease required for 5.8S(S) rRNA synthesis, and, on the other, Rex4p, the RNase III homolog Rnt1p, and the debranching enzyme Dbr1p. Yeast cells carrying a temperature-sensitive mutation in RNase MRP (rrp2-1) exhibit a pre-rRNA processing phenotype very similar to that of the previously studied rrp5-33 mutant: ITS2 processing precedes ITS1 processing, 5.8S(L) rRNA becomes the major species, and ITS1 is processed at the recently reported novel site A4 located midway between sites A2 and A3. As in the rrp5-Δ3 mutant, all of these phenotypical processing features disappear upon inactivation of the REX4 gene. Moreover, inactivation of the DBR1 gene in rrp2-1, or the RNT1 gene in rrp5-Δ3 mutant cells also negates the effects of the original mutation on pre-rRNA processing. These data link a total of three RNA catabolic enzymes, Rex4p, Rnt1p, and Dbr1p, to ITS1 processing and the relative production of 5.8S(S) and 5.8S(L) rRNA. A possible model for the indirect involvement of the three enzymes in yeast pre-rRNA processing is discussed

Proteomic profiling of two distinct populations of extracellular vesicles isolated from human seminal plasma

Body fluids contain many populations of extracellular vesicles (EV) that differ in size, cellular origin, molecular composition, and biological activities. EV in seminal plasma are in majority originating from prostate epithelial cells, and hence are also referred to as prostasomes. Nevertheless, EV are also contributed by other accessory sex glands, as well as by the testis and epididymis. In a previous study, we isolated EV from seminal plasma of vasectomized men, thereby excluding contributions from the testis and epididymis, and identified two distinct EV populations with diameters of 50 and 100 nm, respectively. In the current study, we comprehensively analyzed the protein composition of these two EV populations using quantitative Liquid Chromatography-Mass Spectrometry (LC-MS/MS). In total 1558 proteins were identified. Of these, ≈45% was found only in the isolated 100 nm EV, 1% only in the isolated 50 nm EV, and 54% in both 100 nm and 50 nm EV. Gene ontology (GO) enrichment analysis suggest that both originate from the prostate, but with distinct biogenesis pathways. Finally, nine proteins, including KLK3, KLK2, MSMB, NEFH, PSCA, PABPC1, TGM4, ALOX15B, and ANO7, with known prostate specific expression and alternate expression levels in prostate cancer tissue were identified. These data have potential for the discovery of EV associated prostate cancer biomarkers in blood