A Conditional Yeast E1 Mutant Blocks the Ubiquitin–Proteasome Pathway and Reveals a Role for Ubiquitin Conjugates in Targeting Rad23 to the Proteasome

E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin–proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo

Analysis of 101 nuclear transcriptomes reveals 23 distinct regulons and their relationship to metabolism, chromosomal gene distribution and co-ordination of nuclear and plastid gene expression

Post-endosymbiotic evolution of the proto-chloroplast was characterized by gene transfer to the nucleus. Hence, most chloroplast proteins are nuclear-encoded and the regulation of chloroplast functions includes nuclear transcriptional control. The expression profiles of 3292 nuclear Arabidopsis genes, most of them encoding chloroplast proteins, were determined from 101 different conditions and have been deposited at the GEO database (http://www.ncbi.nlm.nih.gov/geo/) under GSE1160-GSE1260. The 1590 most-regulated genes fell into 23 distinct groups of co-regulated genes (regulons). Genes of some regulons are not evenly distributed among the five Arabidopsis chromosomes and pairs of adjacent, co-expressed genes exist. Except regulons 1 and 2, regulons are heterogeneous and consist of genes coding for proteins with different subcellular locations or contributing to several biochemical functions. This implies that different organelles and/or metabolic pathways are co-ordinated at the nuclear transcriptional level, and a prototype for this is regulon 12 which contains genes with functions in amino acid and carbohydrate metabolism, as well as genes associated with transport or transcription. The co-expression of nuclear genes coding for subunits of the photosystems or encoding proteins involved in the transcription/translation of plastome genes (particularly ribosome polypeptides) (regulons 1 and 2, respectively) implies the existence of a novel mechanism that co-ordinates plastid and nuclear gene expression and involves nuclear control of plastid ribosome abundance. The co-regulation of genes for photosystem and plastid ribosome proteins escapes a previously described general control of nuclear chloroplast proteins imposed by a transcriptional master switch, highlighting a mode of transcriptional regulation of photosynthesis which is different compared to other chloroplast functions. From the evolutionary standpoint, the results provided indicate that functional integration of the proto-chloroplast into the eukaryotic cell was associated with the establishment of different layers of nuclear transcriptional control

Enzymatic Blockade of the Ubiquitin-Proteasome Pathway

Ubiquitin-dependent processes control much of cellular physiology. We show that expression of a highly active, Epstein-Barr virus-derived deubiquitylating enzyme (EBV-DUB) blocks proteasomal degradation of cytosolic and ER-derived proteins by preemptive removal of ubiquitin from proteasome substrates, a treatment less toxic than the use of proteasome inhibitors. Recognition of misfolded proteins in the ER lumen, their dislocation to the cytosol, and degradation are usually tightly coupled but can be uncoupled by the EBV-DUB: a misfolded glycoprotein that originates in the ER accumulates in association with cytosolic chaperones as a deglycosylated intermediate. Our data underscore the necessity of a DUB activity for completion of the dislocation reaction and provide a new means of inhibition of proteasomal proteolysis with reduced cytotoxicity.National Institutes of Health (U.S.)EMBO (long term Fellowship 2008-379)Boehringer Ingelheim Fond

Neuronal inhibition of the autophagy nucleation complex extends life span in post-reproductive C. elegans

Autophagy is a ubiquitous catabolic process that causes cellular bulk degradation of cytoplasmic components and is generally associated with positive effects on health and longevity. Inactivation of autophagy has been linked with detrimental effects on cells and organisms. The antagonistic pleiotropy theory postulates that some fitness-promoting genes during youth are harmful during aging. On this basis, we examined genes mediating post-reproductive longevity using an RNAi screen. From this screen, we identified 30 novel regulators of post-reproductive longevity, including pha-4 Through downstream analysis of pha-4, we identified that the inactivation of genes governing the early stages of autophagy up until the stage of vesicle nucleation, such as bec-1, strongly extend both life span and health span. Furthermore, our data demonstrate that the improvements in health and longevity are mediated through the neurons, resulting in reduced neurodegeneration and sarcopenia. We propose that autophagy switches from advantageous to harmful in the context of an age-associated dysfunction

The inhibition of FGF receptor 1 activity mediates sorafenib-induced antiproliferative effects in human mesothelioma tumor-initiating cells

Tumor-initiating cells (TICs), the subset of cells within tumors endowed with stem-like features, being highly resistant to conventional cytotoxic drugs, are the major cause of tumor relapse. The identification of molecules able to target TICs remains a significant challenge in cancer therapy. Using TIC-enriched cultures (MM1, MM3 and MM4), from 3 human malignant pleural mesotheliomas (MPM), we tested the effects of sorafenib on cell survival and the intracellular mechanisms involved. Sorafenib inhibited cell-cycle progression in all the TIC cultures, but only in MM3 and MM4 cells this effect was associated with induction of apoptosis via the down-regulation of Mcl-1. Although sorafenib inhibits the activity of several tyrosine kinases, its effects are mainly ascribed to Raf inhibition. To investigate the mechanisms of sorafenib-mediated antiproliferative activity, TICs were treated with EGF or bFGF causing, in MM3 and MM4 cells, MEK, ERK1/2, Akt and STAT3 phosphorylation. These effects were significantly reduced by sorafenib in bFGF-treated cells, while a slight inhibition occurred after EGF stimulation, suggesting that sorafenib effects are mainly due to FGFR inhibition. Indeed, FGFR1 phosphorylation was inhibited by sorafenib. A different picture was observed in MM1 cells, which, releasing high levels of bFGF, showed an autocrine activation of FGFR1 and a constitutive phosphorylation/activation of MEK-ERK1/2. A powerful inhibitory response to sorafenib was observed in these cells, indirectly confirming the central role of sorafenib as FGFR inhibitor. These results suggest that bFGF signaling may impact antiproliferative response to sorafenib of MPM TICs, which is mainly mediated by a direct FGFR targeting

E4 ligase–specific ubiquitination hubs coordinate DNA double-strand-break repair and apoptosis

Multiple protein ubiquitination events at DNA double-strand breaks (DSBs) regulate damage recognition, signaling and repair. It has remained poorly understood how the repair process of DSBs is coordinated with the apoptotic response. Here, we identified the E4 ubiquitin ligase UFD-2 as a mediator of DNA-damage-induced apoptosis in a genetic screen in Caenorhabditis elegans. We found that, after initiation of homologous recombination by RAD-51, UFD-2 forms foci that contain substrate-processivity factors including the ubiquitin-selective segregase CDC-48 (p97), the deubiquitination enzyme ATX-3 (Ataxin-3) and the proteasome. In the absence of UFD-2, RAD-51 foci persist, and DNA damage-induced apoptosis is prevented. In contrast, UFD-2 foci are retained until recombination intermediates are removed by the Holliday-junction-processing enzymes GEN-1, MUS-81 or XPF-1. Formation of UFD-2 foci also requires proapoptotic CEP-1 (p53) signaling. Our findings establish a central role of UFD-2 in the coordination between the DNA-repair process and the apoptotic response

Histone H2A Mono-Ubiquitination Is a Crucial Step to Mediate PRC1-Dependent Repression of Developmental Genes to Maintain ES Cell Identity

Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3), respectively. Compared to H3K27me3, localization and the role of H2AK119u1 are not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation

Tebentafusp in Patients with Metastatic Uveal Melanoma: A Real-Life Retrospective Multicenter Study

Background: Tebentafusp has recently been approved for the treatment of metastatic uveal melanoma (mUM) after proving to have survival benefits in a first-line setting. Patients and Methods: This retrospective, multicenter study analyzed the outcomes and safety of tebentafusp therapy in 78 patients with mUM. Results: Patients treated with tebentafusp had a median PFS of 3 months (95% CI 2.7 to 3.3) and a median OS of 22 months (95% CI 10.6 to 33.4). In contrast to a published Phase 3 study, our cohort had a higher rate of patients with elevated LDH (65.4% vs. 35.7%) and included patients with prior systemic and local ablative therapies. In patients treated with tebentafusp following ICI, there was a trend for a longer median OS (28 months, 95% CI 26.9 to 29.1) compared to the inverse treatment sequence (24 months, 95% CI 13.0 to 35.0, p = 0.257). The most common treatment-related adverse events were cytokine release syndrome in 71.2% and skin toxicity in 53.8% of patients. Tumor lysis syndrome occurred in one patient. Conclusions: Data from this real-life cohort showed a median PFS/OS similar to published Phase 3 trial data. Treatment with ICI followed by tebentafusp may result in longer PFS/OS compared to the inverse treatment sequence