Tyrosine Phosphorylation in a Model of Ischemia Using the Rat Hippocampal Slice: Specific, Long-Term Decrease in the Tyrosine Phosphorylation of the Postsynaptic Glycoprotein PSD-GP180

Abstract: The effects of the exposure of hippocampal slices to brief periods of ischemic-like conditions on the tyrosine phosphorylation of proteins and glycoproteins were investigated. Freshly prepared hippocampal slices contained a range of tyrosine-phosphorylated proteins and two prominent tyrosine-phosphorylated glycoproteins of apparent M_r 110,000 (GP110) and 180,000, which we have previously shown to correspond to the postsynaptic density (PSD)-associated glycoprotein PSD-GP180. When hippocampal slices were incubated in oxygenated Krebs-Ringer buffer containing 10 mM glucose (KRB), there was a transient increase in the tyrosine phosphorylation of a protein of M_r 42,000 (p42) and a pronounced increase in the tyrosine phosphorylation of GP110. After these initial changes, the tyrosine phosphorylation of all proteins remained constant for at least 60 min. In vitro “ischemia” was achieved by transferring slices that had been preincubated for 60 min in KRB to KRB that had been equilibrated with N₂ instead of O₂ and that did not contain glucose. Tyrosine-phosphorylated GP110 and PSD-GP180 could no longer be detected after 10 min of exposure of the slices to ischemic-like conditions. GP110 was rapidly rephosphorylated on tyrosine after transfer of slices back to oxygenated, glucose-containing buffer. In contrast, short periods of ischemia (5 or 10 min) resulted in the long-term loss of phosphotyrosine [Tyr(P)]-PSD-GP180 so that it was not detected even after 60 min of reincubation in oxygenated KRB. The sustained decrease in tyrosine phosphorylation of PSD-GP180 after ischemia was Ca²⁺ dependent, the levels of Tyr(P)-PSD-GP180 slowly increasing to preischemic values if Ca²⁺ was omitted from the incubation media. Reoxygenation of ischemic slices also resulted in the Ca²⁺-dependent, transient tyrosine phosphorylation of p42. The major PSD-associated, tyrosine-phosphorylated glycoprotein of molecular mass 180 kDa has recently been identified as the NR2B subunit of the NMDA receptor. The results suggest that changes in tyrosine phosphorylation after an ischemic insult may modulate the NMDA receptor or signal transduction pathways in the postsynaptic cell and are consistent with a role for tyrosine phosphorylation in the sequence of events leading to neuronal cell damage and death.     

DNA demethylases target promoter transposable elements to positively regulate stress responsive genes in Arabidopsis

Background

DNA demethylases regulate DNA methylation levels in eukaryotes. Arabidopsis encodes four DNA demethylases, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), DEMETER-LIKE 2 (DML2), and DML3. While DME is involved in maternal specific gene expression during seed development, the biological function of the remaining DNA demethylases remains unclear.

Results

We show that ROS1, DML2, and DML3 play a role in fungal disease resistance in Arabidopsis. A triple DNA demethylase mutant, rdd (ros1 dml2 dml3), shows increased susceptibility to the fungal pathogen Fusarium oxysporum. We identify 348 genes differentially expressed in rdd relative to wild type, and a significant proportion of these genes are downregulated in rdd and have functions in stress response, suggesting that DNA demethylases maintain or positively regulate the expression of stress response genes required for F. oxysporum resistance. The rdd-downregulated stress response genes are enriched for short transposable element sequences in their promoters. Many of these transposable elements and their surrounding sequences show localized DNA methylation changes in rdd, and a general reduction in CHH methylation, suggesting that RNA-directed DNA methylation (RdDM), responsible for CHH methylation, may participate in DNA demethylase-mediated regulation of stress response genes. Many of the rdd-downregulated stress response genes are downregulated in the RdDM mutants nrpd1 and nrpe1, and the RdDM mutants nrpe1 and ago4 show enhanced susceptibility to F. oxysporum infection.

Conclusions

Our results suggest that a primary function of DNA demethylases in plants is to regulate the expression of stress response genes by targeting promoter transposable element sequences.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0458-3) contains supplementary material, which is available to authorized users.     

Host and Viral Translational Mechanisms during Cricket Paralysis Virus Infection

The dicistrovirus is a positive-strand single-stranded RNA virus that possesses two internal ribosome entry sites (IRES) that direct translation of distinct open reading frames encoding the viral structural and nonstructural proteins. Through an unusual mechanism, the intergenic region (IGR) IRES responsible for viral structural protein expression mimics a tRNA to directly recruit the ribosome and set the ribosome into translational elongation. In this study, we explored the mechanism of host translational shutoff in Drosophila S2 cells infected by the dicistrovirus, cricket paralysis virus (CrPV). CrPV infection of S2 cells results in host translational shutoff concomitant with an increase in viral protein synthesis. CrPV infection resulted in the dissociation of eukaryotic translation initiation factor 4G (eIF4G) and eIF4E early in infection and the induction of deIF2α phosphorylation at 3 h postinfection, which lags after the initial inhibition of host translation. Forced dephosphorylation of deIF2α by overexpression of dGADD34, which activates protein phosphatase I, did not prevent translational shutoff nor alter virus production, demonstrating that deIF2α phosphorylation is dispensable for host translational shutoff. However, premature induction of deIF2α phosphorylation by thapsigargin treatment early in infection reduced viral protein synthesis and replication. Finally, translation mediated by the 5′ untranslated region (5′UTR) and the IGR IRES were resistant to impairment of eIF4F or eIF2 in translation extracts. These results support a model by which the alteration of the deIF4F complex contribute to the shutoff of host translation during CrPV infection, thereby promoting viral protein synthesis via the CrPV 5′UTR and IGR IRES.For productive viral protein expression, viruses have to compete for and hijack the host translational machinery (45). Some viruses such as poliovirus, vesicular stomatitis virus (VSV), and influenza virus selectively antagonize the translation apparatus to shut off host translation, resulting in the release of ribosomes from host mRNAs and the inhibition of antiviral responses. On the other hand, the host cell can counteract through antiviral mechanisms to shutdown viral translation. For instance, viral RNA replication intermediates can trigger PKR, leading to an inhibition of overall translation. To bypass the block in translation, viruses have evolved unique mechanisms to preferentially recruit the ribosome for viral protein synthesis. Thus, the control of the translational machinery during infection is a major focal point in the battle between the host and the virus and often, elucidation of these viral translational shutoff strategies reveals key targets of translational regulation.The majority of cellular mRNAs initiate translation through the recruitment of the cap-binding complex, eukaryotic translation initiation factor 4F (eIF4F), to the 5′ cap of the mRNA (56). eIF4F consists of the cap-binding protein eIF4E, the RNA helicase, eIF4A, and the adaptor protein eIF4G. eIF4G acts as a bridge to join eIF4E and the 40S subunit via eIF3. With the ternary eIF2-Met-tRNA_i-GTP complex bound, the 40S subunit scans in a 5′-to-3′ direction until an AUG start codon is encountered. Here, eIF5 mediates GTP hydrolysis on the ternary complex, releasing the eIFs and subsequently leading to 60S subunit joining to assemble an elongation-competent 80S ribosome. The ternary eIF2-Met-tRNA_i-GTP complex is reactivated for another round of translation by exchange of GDP for GTP, which is mediated by the guanine nucleotide exchange factor, eIF2B. The 3′ poly(A) tail of the mRNA also stimulates translational initiation by binding to the poly(A) binding protein (PABP), which in turn interacts with eIF4G at the 5′end, resulting in a circularized mRNA. PABP has been proposed to enhance eIF4E affinity for the 5′cap and promote 60S joining, indicating that PABP functions at multiple steps of translational initiation (33).A common tactic viruses use to inhibit host translation is to selectively target eIFs. One of the best studied is the cleavage of eIF4G by viral proteases during picornavirus infection. In humans, two isoforms, eIF4GI and eIF4GII, are cleaved early in poliovirus infection by the viral protease 2A, where cleavage of eIFGII correlates more precisely with host translation shutoff (20). Cleavage of eIF4G produces an amino-terminal fragment that binds to eIF4E and a C-terminal fragment that binds to eIF4A and eIF3 (26, 39, 42). PABP is also cleaved by the viral protease 3C during poliovirus infection, thus contributing to shutoff of both host and viral translation and thereby enabling the switch from viral translation to replication (3, 31, 38). Another major target is the availability of the cap-binding protein eIF4E, which is regulated by binding to the repressor protein 4E-BP (21, 41). 4E-BP and eIF4G compete for an overlapping site on eIF4E (42). In its hypophosphorylated state, 4E-BP binds to and sequesters eIF4E, preventing eIF4G recruitment. Dephosphorylation and activation of 4E-BP has been observed during poliovirus, encephalomyocarditis (EMCV), and VSV infections (7, 18).During virus infection, host antiviral responses are triggered that also inhibit translation to counteract viral protein synthesis. An integral antiviral response is phosphorylation at Ser⁵¹ of eIF2α, which reduces the pool of the ternary complex by blocking the eIF2B-dependent exchange of GDP to GTP. In mammals, four known eIF2α kinases exist including the endoplasmic reticulum (ER)-stress-inducible PERK, GCN2, which senses the accumulation of deacylated tRNAs during amino acid starvation conditions; the heme-regulated kinase HRI; and the interferon-inducible double-stranded RNA-binding PKR (64). In mammalian cells, PKR is activated by binding to double-stranded viral RNA replication intermediates, leading to eIF2α phosphorylation and inhibition of overall host and viral translation. PERK and GCN2 have also been shown to be activated during virus infections by VSV and members of the alphavirus family (2, 6, 43, 65, 79). Often, viruses rely on the ER for synthesis and proper folding of viral proteins. The large burden on the ER activates PERK to phosphorylate eIF2α, thereby inhibiting global protein synthesis to reduce the load on the ER (23). Some viruses such as HCV and herpes simplex viruses have adapted to responses that induce eIF2α phosphorylation by producing viral proteins that counteract PKR or modulate the ER stress response (27, 76). Thus, virus infection can trigger several eIF2α kinases that lead to translational shutoff to counteract viral protein synthesis.To circumvent these translation blocks, viruses such as poliovirus and hepatitis C virus utilize internal ribosome entry sites (IRES), which are RNA elements that directly recruit ribosomes in a cap-independent manner and require only a subset of canonical eIFs (15, 25). It is generally thought that IRES-containing viral mRNAs can be translated under conditions when specific eIFs are compromised during infection. Except for a few cases, the specific mechanisms and factors that lead to IRES stimulation is poorly understood. For example, poliovirus and the related EMCV possess an IRES that allows viral translation despite cleavage of eIF4G during infection or inhibiting eIF4E by 4E-BP binding. This type of IRES can still bind to the central domain of eIF4G and mediate 40S subunit recruitment (11, 37, 57).One of the most unique and simplest IRES is found within the intergenic region (IGR) of the Dicistroviridae family (for extensive reviews, see references 28, 36, and 49). Members of this family include the cricket paralysis virus (CrPV), drosophila C virus (DCV), taura syndrome virus, the Plautia stali intestine virus (PSIV), the Rhopalosiphum padi virus (RhPV), and several bee viruses such as the black queen cell virus and the Israeli acute paralysis virus, which has been recently linked to colony collapse disorder (10). The dicistroviruses encode a positive-strand 8- to 10-kb single-stranded RNA genome, which contains two main open reading frames, ORF1 and ORF2, encoding the nonstructural and structural proteins, respectively, separated by an IGR (see Fig. ​Fig.1A).1A). The 5′ end of the CrPV RNA is linked to the viral protein VpG and the 3′ end contains a poly(A) tail (16). Radiolabeling of intracellular RNA in infected cells reveals no subgenomic RNA species smaller than the full-length genomic RNA, and this has been supported by Northern blot analysis (16, 81). Translation of ORF2 is directed by the IGR IRES, whereas ORF1 expression is mediated by an IRES within the 5′ untranslated region (5′UTR) (35, 67, 81, 82). Remarkably, the IGR IRES element can directly recruit the ribosome independently of eIFs or the initiator Met-tRNA_i (29, 30, 54, 80). Furthermore, the IRES occupies the P-site of the ribosome to initiate translation from the ribosomal A-site encoding non-AUG codon (35, 81). Extensive biochemical and structural analyses from several groups have revealed that the IGR IRES mimics a tRNA that occupies the mRNA cleft of the ribosome and sets the ribosome into an elongation state (9, 29, 30, 34, 51, 55, 58, 68, 72, 83). Using reporter constructs, it has also been demonstrated that CrPV IGR IRES-mediated translation is active under a number of cellular conditions when the activity of the ternary complex eIF2-Met-tRNA_i-GTP is compromised (17, 63, 78, 80). Because IGR IRES-mediated translation does not require initiation factors, the IRES can direct translation under a number of cellular conditions when the activity of multiple eIFs is compromised (12). Although the majority of studies have focused on the IGR IRES of CrPV, PSIV, and TSV, it is predicted that the IGRs within this viral family all function similarly based on the predicted conserved RNA structures (28, 36, 49). In contrast, only the 5′UTR IRES mechanism of RhPV has been studied in detail (77). Despite the wealth of studies on the mechanics of these IRES, the mechanisms that lead to translational shutoff during dicistrovirus infection and the interaction of dicistrovirus with the host machinery to allow virus production have been relatively unexplored.Open in a separate windowFIG. 1.Kinetics of host protein synthesis and viral protein expression in CrPV-infected Drosophila S2 cells. (A) Genomic arrangement of the CrPV RNA. The viral open reading frames, ORF1 and ORF2, that encode nonstructural (NS) and structural (S) proteins, respectively, are shown, which are separated by the intergenic internal ribosome entry site (IGR IRES). Translation of ORF1 and ORF2 is directed by the 5′UTR IRES and the IGR IRES, respectively. The first amino acid of ORF2 directed by the IGR IRES is encoded by a GCU alanine codon. (B) Autoradiography of protein lysates resolved on a SDS-12% PAGE gel. The protein lysates were collected from S2 cells that were untreated (U), mock infected (M), CrPV infected (5 FFU/cell), or thapsigargin treated (Tg; 0.4 μM) for the indicated times (h p.i.) and metabolically labeled with [³⁵S]methionine for 30 min at each time point. The migration of proteins with known molecular masses is shown on the left. The expression of detectable nonstructural (NS) and structural (S) proteins is denoted. (C) Quantitation of host protein synthesis during CrPV infection. To calculate the host translation at each time point, the amount of radioactivity of the bands between 55 and 70 kDa in panel A was quantitated by using ImageQuant, and the percent translation was calculated at each time point of virus infection or thapsigargin treatment compared to the mock infection. Shown are averages (± the standard deviation) from at least three independent experiments. (D) Immunoblots of viral ORF1 and ORF2 during CrPV infection at various times postinfection (h p.i.). Antibodies were raised against peptides within ORF1 and ORF2. The expression of ORF1 and ORF2 was quantitated by a LI-COR Odyssey system, plotted against time of infection, and normalized to the amount of ORF1 or ORF2 expression at 6 h p.i. (100%). As a comparison, viral RNA synthesis as detected by Northern blot analysis (see Fig. ​Fig.2B)2B) is plotted on the same graph.Previous studies have shown that the CrPV and the related DCV can infect a wide range of insect hosts, including the Drosophila melanogaster S2 cell line (60, 69). In the present study, we have explored how CrPV infection leads to host translational shutoff in S2 cells. Two steps of translational initiation are targeted during CrPV infection. First, the interaction of deIF4G with deIF4E is disrupted early in infection and remains dissociated during the course of infection. Second, deIF2α is phosphorylated at a time that lags after the initial host translational shutoff during infection. Premature phosphorylation of deIF2α early in infection inhibited translation directed by the 5′UTR IRES, but IGR IRES-mediated translation remained relatively resistant. These results support the model that multiple mechanisms, including impairment of deIF4F complex formation and induction of deIF2α phosphorylation, contribute to the host translational shutoff during CrPV infection. The inhibition of host translation and the release of ribosomes from host mRNAs ensures that translation mediated by the 5′UTR and IGR IRES is optimal to produce sufficient viral nonstructural and structural proteins for proper CrPV maturation and assembly.     

Quantitative proteomics analysis of the secretory pathway

We report more than 1400 proteins of the secretory-pathway proteome and provide spatial information on the relative presence of each protein in the rough and smooth ER Golgi cisternae and Golgi-derived COPI vesicles. The data support a role for COPI vesicles in recycling and cisternal maturation, showing that Golgi-resident proteins are present at a higher concentration than secretory cargo. Of the 1400 proteins, 345 were identified as previously uncharacterized. Of these, 230 had their subcellular location deduced by proteomics. This study provides a comprehensive catalog of the ER and Golgi proteomes with insight into their identity and function.     

Lysine methylation as a routine rescue strategy for protein crystallization

Crystallization remains a critical step in X-ray structure determination. Because it is not generally possible to rationally predict crystallization conditions, commercial screens have been developed which sample a wide range of crystallization space. While this approach has proved successful in many cases, a significant number of proteins fail to crystallize despite being soluble and monodispersed. It is established that chemical modification can facilitate the crystallization of otherwise intractable proteins. Here we describe a method for the reductive methylation of lysine residues which is simple, inexpensive, and efficient, and report on its application to ten proteins. We describe the effect of methylation on the physico-chemical properties of these proteins, and show that it led to diffraction-quality crystals from four proteins and structures for three that had hitherto proved refractory to crystallization. The method is suited to both low- and high-throughput laboratories.     

Chromosomal integration mechanism of infecting mu virion DNA

DNA transposition is central to the propagation of temperate phage Mu. A long-standing problem in Mu biology has been the mechanism by which the linear genome of an infecting phage, which is linked at both ends to DNA acquired from a previous host, integrates into the new host chromosome. If Mu were to use its well-established cointegrate mechanism for integration (single-strand nicks at Mu ends, joined to a staggered double-strand break in the target), the flanking host sequences would remain linked to Mu; target-primed replication of the linear integrant would subsequently break the chromosome. The absence of evidence for chromosome breaks has led to speculation that infecting Mu might use a cut-and-paste mechanism, whereby Mu DNA is cut away from the flanking sequences prior to integration. In this study we have followed the fate of the flanking DNA during the time course of Mu infection. We have found that these sequences are still attached to Mu upon integration and that they disappear soon after. The data rule out a cut-and-paste mechanism and suggest that infecting Mu integrates to generate simple insertions by a variation of its established cointegrate mechanism in which, instead of a "nick, join, and replicate" pathway, it follows a "nick, join, and process" pathway. The results show similarities with human immunodeficiency virus integration and provide a unifying mechanism for development of Mu along either the lysogenic or lytic pathway.     

Stressed-induced TMEM135 protein is part of a conserved genetic network involved in fat storage and longevity regulation in Caenorhabditis elegans

Disorders of mitochondrial fat metabolism lead to sudden death in infants and children. Although survival is possible, the underlying molecular mechanisms which enable this outcome have not yet been clearly identified. Here we describe a conserved genetic network linking disorders of mitochondrial fat metabolism in mice to mechanisms of fat storage and survival in Caenorhabditis elegans (C. elegans). We have previously documented a mouse model of mitochondrial very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency. We originally reported that the mice survived birth, but, upon exposure to cold and fasting stresses, these mice developed cardiac dysfunction, which greatly reduced survival. We used cDNA microarrays to outline the induction of several markers of lipid metabolism in the heart at birth in surviving mice. We hypothesized that the induction of fat metabolism genes in the heart at birth is part of a regulatory feedback circuit that plays a critical role in survival. The present study uses a dual approach employing both C57BL/6 mice and the nematode, C. elegans, to focus on TMEM135, a conserved protein which we have found to be upregulated 4.3 (±0.14)-fold in VLCAD-deficient mice at birth. Our studies have demonstrated that TMEM135 is highly expressed in mitochondria and in fat-loaded tissues in the mouse. Further, when fasting and cold stresses were introduced to mice, we observed 3.25 (±0.03)- and 8.2 (±0.31)-fold increases in TMEM135 expression in the heart, respectively. Additionally, we found that deletion of the tmem135 orthologue in C. elegans caused a 41.8% (±2.8%) reduction in fat stores, a reduction in mitochondrial action potential and decreased longevity of the worm. In stark contrast, C. elegans transgenic animals overexpressing TMEM-135 exhibited increased longevity upon exposure to cold stress. Based on these results, we propose that TMEM135 integrates biological processes involving fat metabolism and energy expenditure in both the worm (invertebrates) and in mammalian organisms. The data obtained from our experiments suggest that TMEM135 is part of a regulatory circuit that plays a critical role in the survival of VLCAD-deficient mice and perhaps in other mitochondrial genetic defects of fat metabolism as well.