Methylglyoxal Alters the Function and Stability of Critical Components of the Protein Quality Control

BACKGROUND: Increased production and accumulation of methylglyoxal (MGO), as well as increased modification of proteins by glycoxidation, are hallmarks of aging and diabetes. MGO was shown to modify proteins and to contribute to the accumulation of damaged proteins that can be toxic to cells. However, the effect of MGO on the cell systems responsible for repairing or degrading damaged proteins is still unclear. In this study, the effect of MGO on the function of the ubiquitin-proteasome system (UPS) and on molecular chaperones, two cooperative mechanisms associated with protein quality control, was investigated. PRINCIPAL FINDINGS: In this work it is shown that treatment of cells with MGO leads to accumulation of ubiquitin conjugates and depletion of free ubiquitin. Moreover, MGO significantly decreases the proteolytic activity of the 20S proteasome. Data further shows that MGO decreases the levels of the molecular chaperones Hsc70 and Hsp90 and leads to accumulation of CHIP-, Hsp40- and ubiquitin-containing aggregates. The formation of large aggregates containing CHIP is a consequence of its binding to misfolded proteins and to molecular chaperones. Moreover, dysfunction of the chaperones/CHIP/UPS axis is associated with accumulation of oxidized and argpyrimidine-modified proteins, which is likely to be associated with decreased cell viability. Interestingly, data further shows that MGO-induced stress induces the activation of heat shock factor-1 (Hsf-1), the main transcription factor involved in the regulation of the expression of heat shock proteins (HSPs) and cell response to stress. CONCLUSIONS: The data obtained in this work suggests that MGO impairs both the UPS and the protein quality control dependent on CHIP and molecular chaperones, leading to accumulation of toxic aggregates and increased cell death. However, these MGO-induced changes appear to elicit a response from the Hsf-1 system, which is crucial to help cells to cope with cellular stress and to re-establish homeostasis

An Interaction Network Predicted from Public Data as a Discovery Tool: Application to the Hsp90 Molecular Chaperone Machine

Understanding the functions of proteins requires information about their protein-protein interactions (PPI). The collective effort of the scientific community generates far more data on any given protein than individual experimental approaches. The latter are often too limited to reveal an interactome comprehensively. We developed a workflow for parallel mining of all major PPI databases, containing data from several model organisms, and to integrate data from the literature for a protein of interest. We applied this novel approach to build the PPI network of the human Hsp90 molecular chaperone machine (Hsp90Int) for which previous efforts have yielded limited and poorly overlapping sets of interactors. We demonstrate the power of the Hsp90Int database as a discovery tool by validating the prediction that the Hsp90 co-chaperone Aha1 is involved in nucleocytoplasmic transport. Thus, we both describe how to build a custom database and introduce a powerful new resource for the scientific community

Detrimental effects of RNAi: a cautionary note on its use in Drosophila ageing studies

RNA interference (RNAi) provides an important tool for gene function discovery. It has been widely exploited in Caenorhabditis elegans ageing research because it does not appear to have any non-specific effects on ageing-related traits in that model organism. We show here that ubiquitous, adult-onset activation of the RNAi machinery, achieved by expressing a double stranded RNA targeting GFP or lacZ for degradation, or by increasing expression of Dicer substantially reduces lifespan in Drosophila melanogaster. Induction of GFPRNAi construct also alters the response of lifespan to nutrition, exacerbating the lifespan-shortening effects of food containing a high quantity of yeast. Our study indicates that activation of the RNAi machinery may have sequence-independent side-effects on lifespan, and that caution needs to be exercised when employing ubiquitous RNAi in Drosophila ageing studies. However, we also show that RNAi restricted to certain tissues may not be detrimental to lifespan

Function of COP9 Signalosome in Regulation of Mouse Oocytes Meiosis by Regulating MPF Activity and Securing Degradation

The COP9 (constitutive photomorphogenic) signalosome (CSN), composed of eight subunits, is a highly conserved protein complex that regulates processes such as cell cycle progression and kinase signalling. Previously, we found the expression of the COP9 constitutive photomorphogenic homolog subunit 3 (CSN3) and subunit 5 (CSN5) changes as oocytes mature for the first time, and there is no report regarding roles of COP9 in the mammalian oocytes. Therefore, in the present study, we examined the effects of RNA interference (RNAi)-mediated transient knockdown of each subunit on the meiotic cell cycle in mice oocytes. Following knockdown of either CSN3 or CSN5, oocytes failed to complete meiosis I. These arrested oocytes exhibited a disrupted meiotic spindle and misarranged chromosomes. Moreover, down-regulation of each subunit disrupted the activity of maturation-promoting factor (MPF) and concurrently reduced degradation of the anaphase-promoting complex/cyclosome (APC/C) substrates Cyclin B1 and Securin. Our data suggest that the CSN3 and CSN5 are involved in oocyte meiosis by regulating degradation of Cyclin B1 and Securin via APC/C

The DNA polymerases of Drosophila melanogaster.

DNA synthesis during replication or repair is a fundamental cellular process that is catalyzed by a set of evolutionary conserved polymerases. Despite a large body of research, the DNA polymerases of Drosophila melanogaster have not yet been systematically reviewed, leading to inconsistencies in their nomenclature, shortcomings in their functional (Gene Ontology, GO) annotations and an under-appreciation of the extent of their characterization. Here, we describe the complete set of DNA polymerases in D. melanogaster, applying nomenclature already in widespread use in other species, and improving their functional annotation. A total of 19 genes encode the proteins comprising three replicative polymerases (alpha-primase, delta, epsilon), five translesion/repair polymerases (zeta, eta, iota, Rev1, theta) and the mitochondrial polymerase (gamma). We also provide an overview of the biochemical and genetic characterization of these factors in D. melanogaster. This work, together with the incorporation of the improved nomenclature and GO annotation into key biological databases, including FlyBase and UniProtKB, will greatly facilitate access to information about these important proteins

Decreased MCM2-6 in Drosophila S2 cells does not generate significant DNA damage or cause a marked increase in sensitivity to replication interference.

A reduction in the level of some MCM proteins in human cancer cells (MCM5 in U20S cells or MCM3 in Hela cells) causes a rapid increase in the level of DNA damage under normal conditions of cell proliferation and a loss of viability when the cells are subjected to replication interference. Here we show that Drosophila S2 cells do not appear to show the same degree of sensitivity to MCM2-6 reduction. Under normal cell growth conditions a reduction of >95% in the levels of MCM3, 5, and 6 causes no significant short term alteration in the parameters of DNA replication or increase in DNA damage. MCM depleted cells challenged with HU do show a decrease in the density of replication forks compared to cells with normal levels of MCM proteins, but this produces no consistent change in the levels of DNA damage observed. In contrast a comparable reduction of MCM7 levels has marked effects on viability, replication parameters and DNA damage in the absence of HU treatment

Nuclear Shield: A Multi-Enzyme Task-Force for Nucleus Protection

In eukaryotic cells the nuclear envelope isolates and protects DNA from molecules that could damage its structure or interfere with its processing. Moreover, selected protection enzymes and vitamins act as efficient guardians against toxic compounds both in the nucleoplasm and in the cytosol. The observation that a cytosolic detoxifying and antioxidant enzyme i.e. glutathione transferase is accumulated in the perinuclear region of the rat hepatocytes suggests that other unrecognized modalities of nuclear protection may exist. Here we show evidence for the existence of a safeguard enzyme machinery formed by an hyper-crowding of cationic enzymes and proteins encompassing the nuclear membrane and promoted by electrostatic interactions

The role of Insulators and transcription in 3D chromatin organisation of flies

The DNA in many organisms, including humans, is shown to be organised in topologically associating domains (TADs). In Drosophila, several architectural proteins are enriched at TAD borders, but it is still unclear whether these proteins play a functional role in the formation and maintenance of TADs. Here, we show that depletion of BEAF-32, Cp190, Chro and Dref leads to changes in TAD organisation and chromatin loops. Their depletion predominantly affects TAD borders located in regions moderately enriched in repressive modifications and depleted in active ones, while TAD borders located in euchromatin are resilient to these knockdowns. Furthermore, transcriptomic data has revealed hundreds of genes displaying differential expression in these knockdowns and showed that the majority of differentially expressed genes are located within reorganised TADs. Our work identifies a novel and functional role for architectural proteins at TAD borders in Drosophila and a link between TAD reorganisation and subsequent changes in gene expression

Fluorescent Protein-Based Methods for On-Plate Screening of Gene Insertion

Unlike the commonly used method of blue-white screening for gene insertion, a fluorescent protein-based screening method offers a gain-of-function screening process without using any co-factors and a gene fusion product with a fluorescent protein reporter that is further useful in cell imaging studies. However, complications related to protein-folding efficiencies of the gene insert in fusion with fluorescent protein reporters prevent effective on-plate bacterial colony selection leading to its limited use.Here, we present three methods to tackle this problem. Our first method promotes the folding of the gene insert by using an N-terminal protein such as calmodulin that is well folded and expressed. Under this method, fluorescence was increased more than 30x over control allowing for enhanced screening. Our second method creates a fluorescent protein that is N-terminal to the gene upon insertion, thereby reducing the dependency of the fluorescent protein reporter on the folding of the gene insert. Our third method eliminates any dependence of the fluorescent protein reporter on the folding of the gene insert by using a stop and start sequence for protein translation.The three methods together will expand the usefulness of fluorescence on-plate screening and offer a powerful alternative to blue-white screening

Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage

Annexin A2 is an abundant cellular protein that is mainly localized in the cytoplasm and plasma membrane, however a small population has been found in the nucleus, suggesting a nuclear function for the protein. Annexin A2 possesses a nuclear export sequence (NES) and inhibition of the NES is sufficient to cause nuclear accumulation. Here we show that annexin A2 accumulates in the nucleus in response to genotoxic agents including gamma-radiation, UV radiation, etoposide and chromium VI and that this event is mediated by the nuclear export sequence of annexin A2. Nuclear accumulation of annexin A2 is blocked by the antioxidant agent N-acetyl cysteine (NAC) and stimulated by hydrogen peroxide (H2O2), suggesting that this is a reactive oxygen species dependent event. In response to genotoxic agents, cells depleted of annexin A2 show enhanced phospho-histone H2AX and p53 levels, increased numbers of p53-binding protein 1 nuclear foci and increased levels of nuclear 8-oxo-2'-deoxyguanine, suggesting that annexin A2 plays a role in protecting DNA from damage. This is the first report showing the nuclear translocation of annexin A2 in response to genotoxic agents and its role in mitigating DNA damage.Natural Sciences and Engineering Research Council of Canada (NSERC); European Union [PCOFUND-GA-2009-246542]; Foundation for Science and Technology of Portugal; Beatrice Hunter Cancer Research Institute; Terry Fox Foundationinfo:eu-repo/semantics/publishedVersio