Familiarity Breeds Strategy: In Silico Untangling of the Molecular Complexity on Course of Autoimmune Liver Disease-to-Hepatocellular Carcinoma Transition Predicts Novel Transcriptional Signatures.

Autoimmune liver diseases (AILD) often lead to transformation of the liver tissues into hepatocellular carcinoma (HCC). Considering the drawbacks of surgical procedures in such cases, need of successful non-invasive therapeutic strategies and treatment modalities for AILD-associated-HCC still exists. Due to the lack of clear, sufficient knowledge about factors mediating AILD-to-HCC transition, an in silico approach was adopted to delineate the underlying molecular deterministic factors. Parallel enrichment analyses on two different public microarray datasets (GSE159676 and GSE62232) pinpointed the core transcriptional regulators as key players. Correlation between the expression kinetics of these transcriptional modules in AILD and HCC was found to be positive primarily with the advancement of hepatic fibrosis. Most of the regulatory interactions were operative during early (F0-F1) and intermediate fibrotic stages (F2-F3), while the extent of activity in the regulatory network considerably diminished at late stage of fibrosis/cirrhosis (F4). Additionally, most of the transcriptional targets with higher degrees of connectivity in the regulatory network (namely DCAF11, PKM2, DGAT2 and BCAT1) may be considered as potential candidates for biomarkers or clinical targets compared to their low-connectivity counterparts. In summary, this study uncovers new possibilities in the designing of novel prognostic and therapeutic regimen for autoimmunity-associated malignancy of liver in a disease progression-dependent fashion.

A new acid mix enhances phosphopeptide enrichment on titanium- and zirconium dioxide for mapping of phosphorylation sites on protein complexes.

The selective enrichment of phosphorylated peptides prior to reversed-phase separation and mass spectrometric detection significantly improves the analytical results in terms of higher number of detected phosphorylation sites and spectra of higher quality. Metal oxide chromatography (MOC) has been recently described for selective phosphopeptide enrichment (Pinkse et al., 2004; Larsen et al., 2005; Kweon and Hakansson, 2006; Cantin et al., 2007; Collins et al., 2007). In the present work we have tested the effect of a modified loading solvent containing a novel acid mix and optimized wash conditions on the efficiency of TiO(2)-based phosphopeptide enrichment in order to improve our previously published method (Mazanek et al., 2007). Applied to a test mixture of synthetic and BSA-derived peptides, the new method showed improved selectivity for phosphopeptides, whilst retaining a high recovery rate. Application of the new enrichment method to digested purified protein complexes resulted in the identification of a significantly higher number of phosphopeptides as compared to the previous method. Additionally, we have compared the performance of TiO(2) and ZrO(2) columns for the isolation and identification of phosphopeptides from purified protein complexes and found that for our test set, both media performed comparably well. In summary, our improved method is highly effective for the enrichment of phosphopeptides from purified protein complexes prior to mass spectrometry, and is suitable for large-scale phosphoproteomic projects that aim to elucidate phosphorylation-dependent cellular processes.

FLEXIQuant: a novel tool for the absolute quantification of proteins, and the simultaneous identification and quantification of potentially modified peptides.

Measurements of protein abundance and quantitative assessment of multiple post-translational modifications (PTMs) within a single protein are increasingly used to understand the control of protein activity, particularly in metazoan cells. General methods of wide applicability and precision/accuracy for quantitative estimation of protein post-translational regulation are lacking. Protein mass spectrometry has evolved from a high-throughput qualitative technique to a potentially general quantitative tool, but there are still serious limitations in dynamic range and coverage. To address some of these limitations, we introduce a novel MS-based quantitative strategy, FLEXIQuant, (Full-Length Expressed Stable Isotope-labeled Proteins for Quantification), which can track changes in relative peptide abundances as a function of PTM, and determine absolute quantity of a protein from its lysate. We examined two subunits of the anaphase-promoting complex, CDC27 and APC5, as a test of our ability to monitor quantitatively, the PTM status of several peptides over time. We find evidence of differential regulation at different sites, a phenomenon we believe will be very widespread. FLEXIQuant proved itself to be capable of serving as a general quantitative tool.

Direct binding to Rsp5 mediates ubiquitin-independent sorting of Sna3 via the multivesicular body pathway.

The sorting of most integral membrane proteins into the lumenal vesicles of multivesicular bodies (MVBs) is dependent on the attachment of ubiquitin (Ub) to their cytosolic domains. However, Ub is not required for sorting of Sna3, an MVB vesicle cargo protein in yeast. We show that Sna3 circumvents Ub-mediated recognition by interacting directly with Rsp5, an E3 Ub ligase that catalyzes monoubiquitination of MVB vesicle cargoes. The PPAY motif in the C-terminal cytosolic domain of Sna3 binds the WW domains in Rsp5, and Sna3 is polyubiquitinated as a consequence of this association. However, Ub does not appear to be required for transport of Sna3 via the MVB pathway because its sorting occurs under conditions in which its ubiquitination is impaired. Consistent with Ub-independent function of the MVB pathway, we show by electron microscopy that the formation of MVB vesicles does not require Rsp5 E3 ligase activity. However, cells expressing a catalytically disabled form of Rsp5 have a greater frequency of smaller MVB vesicles compared with the relatively broad distribution of vesicles seen in MVBs of wild-type cells, suggesting that the formation of MVB vesicles is influenced by Rsp5-mediated ubiquitination.

Characterization of multiple multivesicular body sorting determinants within Sna3: a role for the ubiquitin ligase Rsp5.

A subset of proteins that transit the endosomal system are directed into the intralumenal vesicles of multivesicular bodies (MVBs). MVB formation is critical for a variety of cellular functions including receptor down-regulation, viral budding, antigen presentation, and the generation of lysosome-related organelles. Entry of transmembrane proteins into the intralumenal vesicles of a MVB is a highly regulated process that is positively modulated by covalent modification of cargoes with ubiquitin. To identify additional MVB sorting signals, we examined the previously described ubiquitination-independent MVB cargo Sna3. Although Sna3 ubiquitination is not essential, Sna3 MVB sorting is positively modulated by its ubiquitination. Examination of MVB sorting determinants within a form of Sna3 lacking all lysine residues identified two critical regions: an amino-terminal tyrosine-containing region and a carboxyl-terminal PPAY motif. This PPAY motif interacts with the WW domains of the ubiquitin ligase Rsp5, and mutations in either the WW or, surprisingly, the HECT domains of Rsp5 negatively impacted MVB targeting of lysine-minus Sna3. These data indicate that Rsp5 function is required for MVB targeting of Sna3 in a capacity beyond cargo ubiquitination. These results uncover a series of determinants impacting Sna3 MVB sorting, including unexpected roles for Rsp5.

The proteasome: a novel target for anticancer therapy

The proteasome is an ubiquituous enzyme complexthat plays a critical role in the degradation of manyproteins involved in cell cycle regulation, apoptosisand angiogenesis. Since these pathways are fundamentalfor cell survival and proliferation, particularlyin cancer cells, the inhibition of proteasome is anattractive potential anticancer therapy. Bortezomib(Velcade, formerly PS-341) is an extremely potentand selective proteasome inhibitor that shows strongactivity in in vitro and in vivo laboratory studiesagainst many solid and hematologic tumor types.Moreover, bortezomib, mainly by inhibition of theNF-êB pathway, has a chemosensitizing effect whenadministered together with other antitumoral drugs.Clinical phase I trials, showed good tolerance ofbortezomib at doses that achieved a desired degreeof proteasome inhibition. Phase II studies showedhigh response rates in refractory multiple myelomapatients, which led to the accelerated approval ofbortezomib by the Food and Drug Administration(FDA) and the European Medicines Agency (EMEA)for this indication. A phase III trial comparing bortezomibto dexamethasone in refractory/relapsed multiplemyeloma patients had to be halted due to a survivaladvantage in the bortezomib arm. Additionalstudies are focusing in the potential benefit of bortezomibin newly diagnosed multiple myeloma patients.In other solid and hematological malignancies,phase II studies with bortezomib alone or incombination are ongoing with encouraging results,particularly in lung cancer and lymphoma

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Studies of the ubiquitin proteasome system.

A concept that has arisen over the last decade is that proteins can, in general, be covalently modified by polypeptides, resulting in alterations in their fate and function. The first-identified and most well studied of these modifying polypeptides is ubiquitin. Although targeting for proteasomal degradation is the best studied outcome of ubiquitylation, we now understand that modification of proteins with ubiquitin has numerous other cellular roles that alter protein function and that are unrelated to proteasomal degradation. Ubiquitylation is a complex process that is regulated at the level of both addition and removal of ubiquitin from target proteins. This unit includes a number of different basic protocols that will facilitate the study of components of the ubiquitin system and substrate ubiquitylation both in vitro and in cells. Because another protein modifier, NEDD8, itself regulates aspects of the ubiquitin system, basic protocols on neddylation are also included in this unit.

Expression and assay of HECT domain ligases.

HECT domain ubiquitin ligases (HECT E3s), typified by human E6AP and yeast Rsp5p, are unique among the several classes of known ubiquitin ligases in that they participate directly in the chemistry of substrate ubiquitination reactions. This chapter discusses strategies for the expression of active HECT E3s and the assays that are available for analyzing E2 interaction, ubiquitin-thioester formation, and substrate ubiquitination.

Affinity purification of mitotic anaphase-promoting complex/cyclosome on p13Suc1.

A procedure is described for the affinity purification of the mitotic form of anaphase-promoting complex/cyclosome (APC/C) from HeLa cells. It is based on the binding of mitotically phosphorylated APC/C to the phosphate-binding site of p13(suc1), followed by specific elution with a phosphate-containing compound. The procedure is rapid, simple, and yields 50- to 70-fold purification of soluble APC/C, with a approximately 30% recovery of activity.

Restaging the spindle assembly checkpoint in female mammalian meiosis I.

In mammalian somatic cells, the spindle assembly checkpoint (SAC) is indispensable for ensuring the fidelity of chromosome segregation by delaying cell-cycle progression in the face of even a single misaligned chromosome. In contrast, the role of the SAC in unperturbed mammalian oocytes is less well defined as progression through meiosis I is unaltered in mouse oocytes in the presence of one or a few misaligned chromosomes. Furthermore, attempts to disable the function of the SAC protein, Mad2, in mouse oocytes have produced conflicting results. To gain further insight into SAC function during female mammalian meiosis I, we recently utilised a morpholino-based antisense approach to deplete the majority of Mad2 in mouse oocytes. Our results define a clear role for Mad2 in ensuring the proper timing of meiosis I events and ultimately, in ensuring the fidelity of homologue disjunction. We discuss the implications of these results for the regulation of meiosis I in mammalian oocytes and for the genesis of human aneuploidy.

The anaphase promoting complex/cyclosome is recruited to centromeres by the spindle assembly checkpoint.

The anaphase promoting complex/cyclosome (APC/C) is crucial to the control of cell division (for a review, see ref. 1). It is a multi-subunit ubiquitin ligase that, at defined points during mitosis, targets specific proteins for proteasomal degradation. The APC/C is itself regulated by the spindle or kinetochore checkpoint, which has an important role in maintaining genomic stability by preventing sister chromatid separation until all chromosomes are correctly aligned on the mitotic spindle. The spindle checkpoint regulates the APC/C by inactivating Cdc20, an important co-activator of the APC/C. There is also evidence to indicate that the spindle checkpoint components and Cdc20 are spatially regulated by the mitotic apparatus, in particular they are recruited to improperly attached kinetochores. Here, we show that the APC/C itself co-localizes with components of the spindle checkpoint to improperly attached kinetochores. Indeed, we provide evidence that the spindle checkpoint machinery is required to recruit the APC/C to kinetochores. Our data indicate that the APC/C could be regulated directly by the spindle checkpoint.

Formation and nuclear export of tRNA, rRNA and mRNA is regulated by the ubiquitin ligase Rsp5p.

The yeast ubiquitin-protein ligase Rsp5p regulates processes as diverse as polII transcription and endocytosis. Here, we identify Rsp5p in a screen for tRNA export (tex) mutants. The tex23-1/rsp5-3 mutant, which is complemented by RSP5, not only shows a strong nuclear accumulation of tRNAs at the restrictive temperature, but also is severely impaired in the nuclear export of mRNAs and 60S pre-ribosomal subunits. In contrast, nuclear localization sequence (NLS)-mediated nuclear protein import is unaffected in this mutant. Strikingly, the nuclear RNA export defects seen in the rsp5-3 strain are accompanied by a dramatic inhibition of both rRNA and tRNA processing, a combination of phenotypes that has not been reported for any previously characterized mutation in yeast. These data implicate ubiquitination as a mechanism coordinating the major nuclear RNA biogenesis pathways.