The replisome-coupled E3 ubiquitin ligase Rtt101Mms22 counteracts Mrc1 function to tolerate genotoxic stress

Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101Mms22 ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101Mms22 E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1's replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks.</p

Targeted proteomics reveals compositional dynamics of 60S pre‐ribosomes after nuclear export

Construction and intracellular targeting of eukaryotic pre-ribosomal particles involve a multitude of diverse transiently associating trans-acting assembly factors, energy-consuming enzymes, and transport factors. The ability to rapidly and reliably measure co-enrichment of multiple factors with maturing pre-ribosomal particles presents a major biochemical bottleneck towards revealing their function and the precise contribution of >50 energy-consuming steps that drive ribosome assembly. Here, we devised a workflow that combines genetic trapping, affinity-capture, and selected reaction monitoring mass spectrometry (SRM-MS), to overcome this deficiency. We exploited this approach to interrogate the dynamic proteome of pre-60S particles after nuclear export. We uncovered assembly factors that travel with pre-60S particles to the cytoplasm, where they are released before initiating translation. Notably, we identified a novel shuttling factor that facilitates nuclear export of pre-60S particles. Capturing and quantitating protein interaction networks of trapped intermediates of macromolecular complexes by our workflow is a reliable discovery tool to unveil dynamic processes that contribute to their in vivo assembly and transport

Cannabinoid receptor CB1 mediates baseline and activity-induced survival of new neurons in adult hippocampal neurogenesis

<p>Abstract</p> <p>Background</p> <p>Adult neurogenesis is a particular example of brain plasticity that is partially modulated by the endocannabinoid system. Whereas the impact of synthetic cannabinoids on the neuronal progenitor cells has been described, there has been lack of information about the action of plant-derived extracts on neurogenesis. Therefore we here focused on the effects of Δ9-tetrahydrocannabinol (THC) and Cannabidiol (CBD) fed to female C57Bl/6 and Nestin-GFP-reporter mice on proliferation and maturation of neuronal progenitor cells and spatial learning performance. In addition we used cannabinoid receptor 1 (CB1) deficient mice and treatment with CB1 antagonist AM251 in Nestin-GFP-reporter mice to investigate the role of the CB1 receptor in adult neurogenesis in detail.</p> <p>Results</p> <p>THC and CBD differed in their effects on spatial learning and adult neurogenesis. CBD did not impair learning but increased adult neurogenesis, whereas THC reduced learning without affecting adult neurogenesis. We found the neurogenic effect of CBD to be dependent on the CB1 receptor, which is expressed over the whole dentate gyrus. Similarly, the neurogenic effect of environmental enrichment and voluntary wheel running depends on the presence of the CB1 receptor. We found that in the absence of CB1 receptors, cell proliferation was increased and neuronal differentiation reduced, which could be related to CB1 receptor mediated signaling in Doublecortin (DCX)-expressing intermediate progenitor cells.</p> <p>Conclusion</p> <p>CB1 affected the stages of adult neurogenesis that involve intermediate highly proliferative progenitor cells and the survival and maturation of new neurons. The pro-neurogenic effects of CBD might explain some of the positive therapeutic features of CBD-based compounds.</p

Production of He-4 and (4) in Pb-Pb collisions at root(NN)-N-S=2.76 TeV at the LHC

Results on the production of He-4 and (4) nuclei in Pb-Pb collisions at root(NN)-N-S = 2.76 TeV in the rapidity range vertical bar y vertical bar <1, using the ALICE detector, are presented in this paper. The rapidity densities corresponding to 0-10% central events are found to be dN/dy4(He) = (0.8 +/- 0.4 (stat) +/- 0.3 (syst)) x 10(-6) and dN/dy4 = (1.1 +/- 0.4 (stat) +/- 0.2 (syst)) x 10(-6), respectively. This is in agreement with the statistical thermal model expectation assuming the same chemical freeze-out temperature (T-chem = 156 MeV) as for light hadrons. The measured ratio of (4)/He-4 is 1.4 +/- 0.8 (stat) +/- 0.5 (syst). (C) 2018 Published by Elsevier B.V.Peer reviewe

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

Background Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide.Methods A multimethods analysis was performed as part of the GlobalSurg 3 study-a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital.Findings Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3.85 [95% CI 2.58-5.75]; p&lt;0.0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63.0% vs 82.7%; OR 0.35 [0.23-0.53]; p&lt;0.0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer.Interpretation Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised

Combining data integration and molecular dynamics for target identification in alpha-Synuclein-aggregating neurodegenerative diseases: Structural insights on Synaptojanin-1 (Synj1)

Parkinson's disease (PD), Alzheimer's disease (AD) and Amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases hallmarked by the formation of toxic protein aggregates. However, targeting these aggregates therapeutically have thus far shown no success. The treatment of AD has remained particularly problematic since no new drugs have been approved in the last 15 years. Therefore, novel therapeutic targets need to be identified and explored. Here, through the integration of genomic and proteomic data, a set of proteins with strong links to alpha-synuclein-aggregating neurodegenerative diseases was identified. We propose 17 protein targets that are likely implicated in neurodegeneration and could serve as potential targets. The human phosphatidylinositol 5-phosphatase synaptojanin-1, which has already been independently confirmed to be implicated in Parkinson's and Alzheimer's disease, was among those identified. Despite its involvement in PD and AD, structural aspects are currently missing at the molecular level. We present the first atomistic model of the 5-phosphatase domain of synaptojanin-1 and its binding to its substrate phosphatidylinositol 4,5-bisphosphate (PIP2). We determine structural information on the active site including membrane-embedded molecular dynamics simulations. Deficiency of charge within the active site of the protein is observed, which suggests that a second divalent cation is required to complete dephosphorylation of the substrate. The findings in this work shed light on the protein's binding to phosphatidylinositol 4,5-bisphosphate (PIP2) and give additional insight for future targeting of the protein active site, which might be of interest in neurodegenerative diseases where synaptojanin-1 is overexpressed. (C) 2020 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology.ISSN:2001-037

Targeted proteomics reveals compositional dynamics of 60S pre-ribosomes after nuclear export

Construction and intracellular targeting of eukaryotic pre‐ribosomal particles involve a multitude of diverse transiently associating trans‐acting assembly factors, energy‐consuming enzymes, and transport factors. The ability to rapidly and reliably measure co‐enrichment of multiple factors with maturing pre‐ribosomal particles presents a major biochemical bottleneck towards revealing their function and the precise contribution of >50 energy‐consuming steps that drive ribosome assembly. Here, we devised a workflow that combines genetic trapping, affinity‐capture, and selected reaction monitoring mass spectrometry (SRM‐MS), to overcome this deficiency. We exploited this approach to interrogate the dynamic proteome of pre‐60S particles after nuclear export. We uncovered assembly factors that travel with pre‐60S particles to the cytoplasm, where they are released before initiating translation. Notably, we identified a novel shuttling factor that facilitates nuclear export of pre‐60S particles. Capturing and quantitating protein interaction networks of trapped intermediates of macromolecular complexes by our workflow is a reliable discovery tool to unveil dynamic processes that contribute to their in vivo assembly and transport.ISSN:1744-429

A sentinel protein assay for simultaneously quantifying cellular processes

We describe a proteomic screening approach based on the concept of sentinel proteins, biological markers whose change in abundance characterizes the activation state of a given cellular process. Our sentinel assay simultaneously probed 188 biological processes in Saccharomyces cerevisiae exposed to a set of environmental perturbations. The approach can be applied to analyze responses to large sets of uncharacterized perturbations in high throughput