20 research outputs found

    Toward accurate reconstruction of functional protein networks

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    Genome-scale screening studies are gradually accumulating a wealth of data on the putative involvement of hundreds of genes/proteins in various cellular responses or functions. A fundamental challenge is to chart out the protein pathways that underlie these systems. Previous approaches to the problem have either employed a local optimization criterion, aiming to infer each pathway independently, or a global criterion, searching for the overall most parsimonious subnetwork. Here, we study the trade-off between the two approaches and present a new intermediary scheme that provides explicit control over it. We demonstrate its utility in the analysis of the apoptosis network in humans, and the telomere length maintenance (TLM) system in yeast. Our results show that in the majority of real-life cases, the intermediary approach provides the most plausible solutions. We use a new set of perturbation experiments measuring the role of essential genes in telomere length regulation to further study the TLM network. Surprisingly, we find that the proteasome plays an important role in telomere length regulation through its associations with transcription and DNA repair circuits

    Peroxisomes:New insights into protein sorting, dynamics, quality control, signalling and roles in health and disease

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    The 6th Open European peroxisome meeting (OEPM) was held on the 26th and 27th of October (2018) in Groningen, the Netherlands. OEPM is a biannual meeting organized by a European peroxisome research group. Previous meetings were held in Leuven, BE (2006), Lunteren, NL (2010), Dijon, FR (2012), Neuss, GER (2014) and Vienna, AU (2016). Over 120 participants were registered from 14 European countries, as well as Israel, Canada, the USA and South Korea. A large number of European research groups participated, including established and younger groups, showing that peroxisome research is blooming in Europe. This will further expand with the EU Marie Curie Innovative training network PERICO (PERoxisome Interactions and COmmunication; http://www.itn-PERICO.eu; coordinated by Ida van der Klei), which recently started and aims to train the next generation of peroxisome researchers

    Validation of a yeast malate dehydrogenase 2 (Mdh2) antibody tested for use in western blots [version 1; referees: 2 approved]

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    Malate dehydrogenases (Mdhs) reversibly convert malate to oxaloacetate and serve as important enzymes in several metabolic pathways. In the yeast Saccharomyces cerevisiae there are three Mdh isozymes, localized to different compartments in the cell. In order to identify specifically the Mdh2 isozyme, GenScript USA produced three different antibodies that we further tested by western blot. All three antibodies recognized the S. cerevisiae Mdh2 with different background and specificity properties. One of the antibodies had a relatively low background and high specificity and thus can be used for specific identification of Mdh2 in various experimental settings

    Nucleosome maps of the human cytomegalovirus genome reveal a temporal switch in chromatin organization linked to a major IE protein

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    Human CMV (hCMV) establishes lifelong infections in most of us, causing developmental defects in human embryos and life-threatening disease in immunocompromised individuals. During productive infection, the viral >230,000-bp dsDNA genome is expressed widely and in a temporal cascade. The hCMV genome does not carry histones when encapsidated but has been proposed to form nucleosomes after release into the host cell nucleus. Here, we present hCMV genome-wide nucleosome occupancy and nascent transcript maps during infection of permissive human primary cells. We show that nucleosomes occupy nuclear viral DNA in a nonrandom and highly predictable fashion. At early times of infection, nucleosomes associate with the hCMV genome largely according to their intrinsic DNA sequence preferences, indicating that initial nucleosome formation is genetically encoded in the virus. However, as infection proceeds to the late phase, nucleosomes redistribute extensively to establish patterns mostly determined by nongenetic factors. We propose that these factors include key regulators of viral gene expression encoded at the hCMV major immediate-early (IE) locus. Indeed, mutant virus genomes deficient for IE1 expression exhibit globally increased nucleosome loads and reduced nucleosome dynamics compared with WT genomes. The temporal nucleosome occupancy differences between IE1-deficient and WT viruses correlate inversely with changes in the pattern of viral nascent and total transcript accumulation. These results provide a framework of spatial and temporal nucleosome organization across the genome of a major human pathogen and suggest that an hCMV major IE protein governs overall viral chromatin structure and function

    Novel targeting assay uncovers targeting information within peroxisomal ABC transporter Pxa1

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    The mechanism behind peroxisomal membrane protein targeting is still poorly understood, with only two yeast proteins believed to be involved and no consensus targeting sequence. Pex19 is thought to bind peroxisomal membrane proteins in the cytosol, and is subsequently recruited by Pex3 at the peroxisomal surface, followed by protein insertion via a mechanism that is as-yet-unknown. However, some peroxisomal membrane proteins still correctly sort in the absence of Pex3 or Pex19, suggesting that multiple sorting pathways exist. Here, we studied sorting of yeast peroxisomal ABC transporter Pxa1. Co-localisation analysis of Pxa1-GFP in a collection of 86 peroxisome-related deletion strains revealed that Pxa1 sorting requires Pex3 and Pex19, while none of the other 84 proteins tested were essential. To identify regions with peroxisomal targeting information in Pxa1, we developed a novel in vivo re-targeting assay, using a reporter consisting of the mitochondrial ABC transporter Mdl1 lacking its N-terminal mitochondrial targeting signal. Using this assay, we showed that the N-terminal 95 residues of Pxa1 are sufficient for retargeting this reporter to peroxisomes. Interestingly, truncated Pxa1 lacking residues 1–95 still localised to peroxisomes. This was confirmed via localisation of various Pxa1 truncation and deletion constructs. However, localisation of Pxa1 lacking residues 1–95 depended on the presence of its interaction partner Pxa2, indicating that this truncated protein does not contain a true targeting signal

    Arf1 coordinates fatty acid metabolism and mitochondrial homeostasis

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    Lipid mobilization through fatty acid β-oxidation is a central process essential for energy production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where β-oxidation products enter mitochondria and fuel the tricarboxylic acid cycle. Little is known about the physical and metabolic cooperation between these organelles. Here we found that expression of fatty acid transporters and of the rate-limiting enzyme involved in β-oxidation is decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. Consequently, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although β-oxidation occurs in both mitochondria and peroxisomes in mammals, Arf1's role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact sites
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