Solution Structure of the SGTA Dimerisation Domain and Investigation of Its Interactions with the Ubiquitin-Like Domains of BAG6 and UBL4A

Background

The BAG6 complex resides in the cytosol and acts as a sorting point to target diverse hydrophobic protein substrates along their appropriate paths, including proteasomal degradation and ER membrane insertion. Composed of a trimeric complex of BAG6, TRC35 and UBL4A, the BAG6 complex is closely associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates.

Methodology and Principal Findings

SGTA consists of an N-terminal dimerisation domain (SGTA_NT), a central tetratricopeptide repeat (TPR) domain, and a glutamine rich region towards the C-terminus. Here we solve a solution structure of the SGTA dimerisation domain and use biophysical techniques to investigate its interaction with two different UBL domains from the BAG6 complex. The SGTA_NT structure is a dimer with a tight hydrophobic interface connecting two sets of four alpha helices. Using a combination of NMR chemical shift perturbation, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) experiments we have biochemically characterised the interactions of SGTA with components of the BAG6 complex, the ubiquitin-like domain (UBL) containing proteins UBL4A and BAG6. We demonstrate that the UBL domains from UBL4A and BAG6 directly compete for binding to SGTA at the same site. Using a combination of structural and interaction data we have implemented the HADDOCK protein-protein interaction docking tool to generate models of the SGTA-UBL complexes.

Significance

This atomic level information contributes to our understanding of the way in which hydrophobic proteins have their fate decided by the collaboration between SGTA and the BAG6 complex.     

GST pull-down联合质谱鉴定BAG结构域相互作用蛋白

目的:BAG结构域(BAG domain,BD)为BAG家族蛋白的基本功能结构域,通过对BAG家族蛋白6个成员的9个BDs的相互作用蛋白进行分析,以探明不同BD相互作用蛋白的异同点并为研究BAG家族蛋白多样性生物功能的分子机制提供理论依据。方法:构建p-GEX-4T2-BDs重组子并转化E.coli BL21(DE3)经IPTG诱导表达GST-BDs融合蛋白并纯化。采用GST pulldown技术联合高效液相色谱串联质谱(LC-MS/MS)的策略对BDs相互作用蛋白进行定性定量分析。最后,用DAVID(The Database for Annotation,Visualization and Intergrated Discovery)和cytoscape对BDs相互作用蛋白进行GO(Gene Ontology)功能分析及KEGG(Kyoto Enyoolpedia of Genes and Genomes)通路分析。结果:在Hela细胞的胞浆蛋白中总共鉴定到370个潜在的BDs相互作用蛋白,主要为核糖体蛋白(ribosomal proteins)、翻译起始因子(Eukaryotic translation initiation factors)、翻译延长因子(Eukaryotic translation elongation factors)、泛素化-蛋白酶体相关蛋白(ubiquitin-proteasome associated proteins)及HSP40家族蛋白。GO功能富集分析结果显示,BDs相互作用蛋白涉及多种生物学功能,包括细胞内蛋白质质量控制(protein quality control)、糖代谢(glycolysis)、免疫调控(immune response)、应激反应(stress response)、细胞周期(cell cycle)等。KEGG通路分析结果表明BDs相互作用蛋白参与多条细胞内重要的信号通路,包括FGF信号通路(FGF signaling pathway)、EGF受体信号通路(EGF receptor signaling pathway)、PDGF信号通路(PDGF signaling pathway)、Ras通路(Ras pathway)等。结论:BAG家族蛋白不同成员的BD所介导的蛋白-蛋白相互作用既有共性又有特异性,BAG家族蛋白通过BDs介导多种蛋白相互作用并参与细胞内多条重要的信号通路来调控细胞内蛋白质稳态、糖代谢、免疫反应、应激反应、细胞周期等过程。     

Endoplasmic Reticulum Targeting and Insertion of Tail-Anchored Membrane Proteins by the GET Pathway

Hundreds of eukaryotic membrane proteins are anchored to membranes by a single transmembrane domain at their carboxyl terminus. Many of these tail-anchored (TA) proteins are posttranslationally targeted to the endoplasmic reticulum (ER) membrane for insertion by the guided-entry of TA protein insertion (GET) pathway. In recent years, most of the components of this conserved pathway have been biochemically and structurally characterized. Get3 is the pathway-targeting factor that uses nucleotide-linked conformational changes to mediate the delivery of TA proteins between the GET pretargeting machinery in the cytosol and the transmembrane pathway components in the ER. Here we focus on the mechanism of the yeast GET pathway and make a speculative analogy between its membrane insertion step and the ATPase-driven cycle of ABC transporters.The mechanism of membrane protein insertion into the endoplasmic reticulum (ER) has been extensively studied for many years (Shao and Hegde 2011). From this work, the signal recognition particle (SRP)/Sec61 pathway has emerged as a textbook example of a cotranslational membrane insertion mechanism (Grudnik et al. 2009). The SRP binds a hydrophobic segment (either a cleavable amino-terminal signal sequence or a transmembrane domain) immediately after it emerges from the ribosomal exit tunnel. This results in a translational pause that persists until SRP engages its receptor in the ER and delivers the ribosome-nascent chain complex to the Sec61 channel. Last, the Sec61 channel enables protein translocation into the ER lumen along with partitioning of hydrophobic transmembrane domains into the lipid bilayer through the Sec61 lateral gate (Rapoport 2007).Approximately 5% of all eukaryotic membrane proteins have an ER targeting signal in a single carboxy-terminal transmembrane domain that emerges from the ribosome exit tunnel following completion of protein synthesis and is not recognized by the SRP (Stefanovic and Hegde 2007). Nonetheless, because hydrophobic peptides in the cytoplasm are prone to aggregation and subject to degradation by quality control systems (Hessa et al. 2011), these tail-anchored (TA) proteins still have to be specifically recognized, shielded from the aqueous environment, and guided to the ER membrane for insertion. In the past five years, the guided-entry of TA proteins (GET) pathway has come to prominence as the major machinery for performing these tasks and the enabler of many key cellular processes mediated by TA proteins including vesicle fusion, membrane protein insertion, and apoptosis. This research has rapidly yielded biochemical and structural insights (​and2)2) into many of the GET pathway components (Hegde and Keenan 2011; Chartron et al. 2012a; Denic 2012). In particular, Get3 is an ATPase that uses metabolic energy to bridge recognition of TA proteins by upstream pathway components with TA protein recruitment to the ER for membrane insertion. However, the precise mechanisms of nucleotide-dependent TA protein binding to Get3 and how the GET pathway inserts tail anchors into the membrane are still poorly understood. Here, we provide an overview of the budding yeast GET pathway with emphasis on mechanistic insights that have come from structural studies of its membrane-associated steps and make a speculative juxtaposition with the ABC transporter mechanism.

Table 1.

A catalog of GET pathway component structures

Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope

Background

Tail-anchored (TA) proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope.

Methodology/Principal Findings

Here we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34) and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9). Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein.

Conclusions/Significance

Collectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie the delivery of TA proteins to their proper intracellular locations in general.     

Association of Fission Yeast Orp1 and Mcm6 Proteins with Chromosomal Replication Origins

We have previously shown that replication of fission yeast chromosomes is initiated in distinct regions. Analyses of autonomous replicating sequences have suggested that regions required for replication are very different from those in budding yeast. Here, we present evidence that fission yeast replication origins are specifically associated with proteins that participate in initiation of replication. Most Orp1p, a putative subunit of the fission yeast origin recognition complex (ORC), was found to be associated with chromatin-enriched insoluble components throughout the cell cycle. In contrast, the minichromosome maintenance (Mcm) proteins, SpMcm2p and SpMcm6p, encoded by the nda1(+)/cdc19(+) and mis5(+) genes, respectively, were associated with chromatin DNA only during the G(1) and S phases. Immunostaining of spread nuclei showed SpMcm6p to be localized at discrete foci on chromatin during the G(1) and S phases. A chromatin immunoprecipitation assay demonstrated that Orp1p was preferentially localized at the ars2004 and ars3002 origins of the chromosome throughout the cell cycle, while SpMcm6p was associated with these origins only in the G(1) and S phases. Both Orp1p and SpMcm6p were associated with a 1-kb region that contains elements required for autonomous replication of ars2004. The results suggest that the fission yeast ORC specifically interacts with chromosomal replication origins and that Mcm proteins are loaded onto the origins to play a role in initiation of replication.     

Association of the P6 Protein of Cauliflower mosaic virus with Plasmodesmata and Plasmodesmal Proteins

The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata.Through the years, numerous studies have focused on the characterization of viral replication sites within the cell, as well as how plant virus movement proteins (MPs) modify the plasmodesmata to facilitate cell-to-cell movement (for review, see Benitez-Alfonso et al., 2010; Laliberté and Sanfaçon, 2010; Niehl and Heinlein, 2011; Ueki and Citovsky, 2011; Verchot, 2012). It is accepted that plant virus replication is associated with host membranes, and at some point, the viral genomic nucleic acid must be transferred from the site of replication in the cell to the plasmodesmata. This step could involve transport from a distant site within the cell, or alternatively, it may be that replication is coupled with transport at the entrance of the plasmodesmata (Tilsner et al., 2013). However, even with the latter model, there is ample evidence that the viral proteins necessary for replication or cell-to-cell movement utilize intracellular trafficking pathways within the cell to become positioned at the plasmodesma. These pathways may involve microfilaments, microtubules, or specific endomembranes that participate in macromolecular transport pathways, or combinations of these elements (Harries et al., 2010; Schoelz et al., 2011; Patarroyo et al., 2012; Peña and Heinlein, 2012; Tilsner and Oparka 2012; Liu and Nelson, 2013).The P6 protein of Cauliflower mosaic virus (CaMV) is one viral protein that had not been considered to play a role in viral movement until recently. P6 is the most abundant protein component of the amorphous, electron-dense inclusion bodies (IBs) present during virus infection (Odell and Howell, 1980; Shockey et al., 1980). Ectopic expression of P6 in Nicotiana benthamiana leaves resulted in the formation of inclusion-like bodies (I-LBs) that were capable of intracellular movement along actin microfilaments. Furthermore, treatment of Nicotiana edwardsonii leaves with latrunculin B abolished the formation of CaMV local lesions, suggesting that intact microfilaments are required for CaMV infection (Harries et al., 2009a). A subsequent paper showed that P6 physically interacts with Chloroplast Unusual Positioning1 (CHUP1), a plant protein localized to the chloroplast outer membrane that contributes to movement of chloroplasts on microfilaments in response to changes in light intensity (Oikawa et al., 2003, 2008; Angel et al., 2013). The implication was that P6 might hijack CHUP1 to facilitate movement of the P6 IBs on microfilaments. Silencing of CHUP1 in N. edwardsonii, a host for CaMV, slowed the rate of local lesion formation, suggesting that CHUP1 contributes to intracellular movement of CaMV (Angel et al., 2013).In addition to its role in intracellular trafficking, the P6 protein has been shown to have at least four other distinct functions in the viral infection cycle. P6-containing IBs induced during virus infection are likely virion factories, as they are the primary site for CaMV protein synthesis, genome replication, and assembly of virions (Hohn and Fütterer, 1997). Second, P6 interacts with host ribosomes to facilitate reinitiation of translation of genes on the polycistronic 35S viral RNA, a process called translational transactivation (Bonneville et al., 1989; Park et al., 2001; Ryabova et al., 2002). The translational transactivator region of P6 (Fig. 1) defines the essential sequences required for translational transactivation (DeTapia et al., 1993). Third, P6 is an important pathogenicity determinant. P6 functions as an avirulence determinant in some solanaceous and cruciferous species (Daubert et al., 1984; Schoelz et al., 1986; Hapiak et al., 2008) and is a chlorosis symptom determinant in susceptible hosts (Daubert et al., 1984; Baughman et al., 1988; Goldberg et al., 1991; Cecchini et al., 1997). Finally, P6 has the capacity to compromise host defenses, as it is a suppressor of RNA silencing and cell death (Love et al., 2007; Haas et al., 2008), and it modulates signaling by salicylic acid, jasmonic acid, ethylene, and auxin (Geri et al., 2004; Love et al., 2012; Laird et al., 2013). Domain D1 of P6 has been shown to be necessary but not sufficient for suppression of silencing and salicylic acid-mediated defenses (Laird et al., 2013).Open in a separate windowFigure 1.CaMV and host constructs used for confocal microscopy or coimmunoprecipitation (co-IP). A, Structure of CaMV P6 and Arabidopsis (Arabidopsis thaliana) Soybean Response to Cold (AtSRC2.2) proteins. The functions of P6 domains D1 to D4 tested for interaction with AtSRC2.2 are indicated by the shaded boxes. The Mini TAV is the minimal region for the translational transactivation function. The NLSa sequence corresponds to the nuclear localization signal of influenza virus. The NLS sequence corresponds to the nuclear localization signal of human ribosomal protein L22. B, Structure of P6 (Angel et al., 2013), AtSRC2.2, PDLP (Thomas et al., 2008), and CaMV MP fusions developed for confocal microscopy and/or co-IP. aa, Amino acid.Because P6-containing IBs are the site for virion accumulation and they are capable of movement, they may be responsible for delivering virions to the CaMV MP located at the plasmodesmata (for review, see Schoelz et al., 2011). The vast majority of CaMV virions accumulate in association with P6-containing IBs. Furthermore, P6 physically interacts with the CaMV capsid and MP, as well as the two proteins necessary for aphid transmission, P2 and P3 (Himmelbach et al., 1996; Ryabova et al., 2002; Hapiak et al., 2008; Lutz et al., 2012). Recent studies have indicated that P6 IBs serve as a reservoir for virions, in which the virions may be rapidly transferred to P2 electron-lucent IBs for acquisition by aphids (Bak et al., 2013). It stands to reason that P6 IBs may also serve as a reservoir for CaMV virions to be transferred to the CaMV MP in the plasmodesmata.CaMV virions move from cell to cell through plasmodesmata modified into tubules through the function of its MP (Perbal et al., 1993; Kasteel et al., 1996). However, studies have suggested that CaMV virions do not appear to directly interact with the MP. Instead, the MP interacts with the CaMV P3 protein (also known as the virion-associated protein [VAP]), which forms a trimeric structure that is anchored into the virions (Leclerc et al., 1998; Leclerc et al., 2001). Electron microscopy studies have indicated that MP and VAP colocalize with virions only at the entrance to or within the plasmodesmata, and it has been suggested that the VAP/virion complex travels to the plasmodesmata independently from the MP (Stavolone et al., 2005). Consequently, there is a need for a second CaMV protein such as P6 to fulfill the role of delivery of virions to the plasmodesmata (Schoelz et al., 2011).Additional studies have shown that the CaMV MP is incorporated into vesicles and is trafficked on the endomembrane system to reach the plasmodesma (Carluccio et al., 2014). These authors suggest that the CaMV MP is recycled in a vesicular transport pathway between plasmodesmata and early endosome compartments. The CaMV MP interacts with µA-Adaptin (Carluccio et al., 2014) and Movement Protein-Interacting7 (Huang et al., 2001), two proteins shown to have a role in vesicular trafficking. Once the MP arrives at plasmodesmata, it interacts with the Plasmodesmata-Localized Protein (PDLP) proteins, which comprise a family of eight proteins associated with plasmodesmata (Amari et al., 2010). In addition to its interaction with CaMV MP, PDLP1 interacts with the 2B protein of Grapevine fan leaf virus (GFLV) at the base of tubules formed by the 2B protein. Furthermore, an Arabidopsis transfer DNA (T-DNA) mutant line in which three PDLP genes had been knocked out (pdlp1-pdlp2-pdlp3) responded to GFLV and CaMV inoculation with a delayed infection (Amari et al., 2010). This has led to the suggestion that the PDLPs might act as receptors for the MPs of the tubule-forming viruses such as GFLV and CaMV (Amari et al., 2010, 2011).To better understand the function of the P6 protein during CaMV intracellular movement, we have utilized a yeast (Saccharomyces cerevisiae) two-hybrid assay to identify host proteins that interact with CaMV P6. We show that P6 physically interacts with a C2-calcium-dependent protein (designated AtSRC2.2). AtSRC2.2 is a membrane-bound protein that is capable of forming punctate spots associated with plasmodesmata. The localization of AtSRC2.2 with plasmodesmata led to an analysis of interactions between P6 I-LBs, AtSRC2.2, PDLP1, and the CaMV MP and also revealed that a portion of P6 I-LBs are found adjacent to plasmodesmata. These results provide further evidence for a model in which P6 IBs are capable of delivery of virions to plasmodesmata for their transit to other host cells.     

The Association of Tau-Like Proteins with Vimentin Filaments in Cultured Cells

There is increasing evidence that the different polymers that constitute the cytoskeleton are interconnected to form a three-dimensional network. The macromolecular interaction patterns that stabilize this network and its intrinsic dynamics are the basis for numerous cellular processes. Within this context,in vitrostudies have pointed to the existence of specific associations between microtubules, microfilaments, and intermediate filaments. It has also been postulated that microtubule-associated proteins (MAPs) are directly involved in mediating these interactions. The interactions of tau with vimentin filaments, and its relationships with other filaments of the cytoskeletal network, were analyzed in SW-13 adenocarcinoma cells, through an integrated approach that included biochemical and immunological studies. This cell line has the advantage of presenting a wild-type clone (vim+) and a mutant clone (vim−) which is deficient in vimentin expression. We analyzed the cellular roles of tau, focusing on its interactions with vimentin filaments, within the context of its functional aspects in the organization of the cytoskeletal network. Cosedimentation experiments of microtubular protein with vimentin in cell extracts enriched in intermediate filaments, combined with studies on the direct interaction of tau with nitrocellulose-bound vimentin and analysis of tau binding to vimentin immobilized in single-strand DNA affinity columns, indicate that tau interacts with the vimentin network. These studies were confirmed by a quantitative analysis of the immunofluorescence patterns of cytoskeleton-associated tubulin, tau, and vimentin using flow cytometry. In this regard, a decrease in the levels of tau associated to the cytoskeletal network in the vim− cell mutant compared with the wild-type clones was observed. However, immunofluorescence data on SW-13 cells suggest that the absence of a structured network of vimentin in the mutant vim− cells does not affect the cytoplasmic organization formed by microtubules and actin filaments, when compared with the wild-type vim+ cells. These studies suggest that tau associates with vimentin filaments and that these interactions may play a structural role in cells containing these filaments.     

Structural and Functional Characterization of Ybr137wp Implicates Its Involvement in the Targeting of Tail-Anchored Proteins to Membranes

Nearly 5% of membrane proteins are guided to nuclear, endoplasmic reticulum (ER), mitochondrial, Golgi, or peroxisome membranes by their C-terminal transmembrane domain and are classified as tail-anchored (TA) membrane proteins. In Saccharomyces cerevisiae, the guided entry of TA protein (GET) pathway has been shown to function in the delivery of TA proteins to the ER. The sorting complex for this pathway is comprised of Sgt2, Get4, and Get5 and facilitates the loading of nascent tail-anchored proteins onto the Get3 ATPase. Multiple pulldown assays also indicated that Ybr137wp associates with this complex in vivo. Here, we report a 2.8-Å-resolution crystal structure for Ybr137wp from Saccharomyces cerevisiae. The protein is a decamer in the crystal and also in solution, as observed by size exclusion chromatography and analytical ultracentrifugation. In addition, isothermal titration calorimetry indicated that the C-terminal acidic motif of Ybr137wp interacts with the tetratricopeptide repeat (TPR) domain of Sgt2. Moreover, an in vivo study demonstrated that Ybr137wp is induced in yeast exiting the log phase and ameliorates the defect of TA protein delivery and cell viability derived by the impaired GET system under starvation conditions. Therefore, this study suggests a possible role for Ybr137wp related to targeting of tail-anchored proteins.     

The Association of "Pathogenesis-related" Proteins with Viral Induced Necrosis in Nicotiana sylvestris

The association of “pathogenesis-related” (PR) proteins with protection from superinfection, systemic acquired resistance and production of localized necrotic lesions was examined with a system using tobacco mosaic virus (TMV) and Nicotiana sylvestris. Leaves of N. sylvestris with a mosaic from earlier inoculation with a systemically infecting strain of TMV (TMV-C) and control plants were challenged with a necrotizing strain of TMV (TMV-P), RNA of TMV-P and turnip mosaic virus (TuMV). TMV-P virions produced localized necrotic lesions only in the dark green areas of the mosaic of TMV-C infected plants. Both RNA of TMV-P and TuMV produced localized necrotic lesions in both light green and dark green areas of the mosaic of TMV-C infected plants. All three challenge inocula produced localized necrotic lesions in previously uninoculated plants. Six days after challenge inoculation proteins were extracted from separated dark green and light green mosaic leaf tissue, and leaf material from control plants. Proteins were separated by electrophoresis in a 5 % polyacrylamide spacer gel and 10 % polyacrylamide running gel. PR proteins were found in tissue where localized necrotic lesions were produced as a result of challenge inoculation, but not in tissue that was not superinfected. PR proteins were not found in light green or dark green mosaic leaf tissue as a result of TMV-C inoculation. No PR proteins were evident in protein extracts from light green tissue challenged with TMV-P, although PR proteins were produced in dark green tissue, where necrosis occurred, from the same leaves. Systemic acquired resistance (reduction in size of lesions formed by a challenge inoculation) to TuMV or RNA of TMV-P and PR protein concentration was measured at various times in light green areas of mosaic leaves where dark green areas of the mosaic leaves had been inoculated with TMV-P. No quantitative or temporal relationship between the onset of resistance and PR protein production was found. It is concluded that PR proteins are a result of pathogen induced necrosis and not significantly involved in the mechanism(s) of viral induced resistance.     

Association of the FAM46A Gene VNTRs and BAG6 rs3117582 SNP with Non Small Cell Lung Cancer (NSCLC) in Croatian and Norwegian Populations

We analyzed for associations between a variable number of tandem repeat (VNTR) polymorphism in the Family with sequence similarity 46, member A (FAM46A) gene and a single nucleotide polymorphism (rs3117582) in the BCL2-Associated Athanogene 6 (BAG6) with non small cell lung cancer in Croatian and Norwegian subjects. A total of 503 (262 Croatian and 241Norwegian) non small cell lung cancer patients and 897 controls (568 Croatian and 329 Norwegian) were analyzed. We found that the frequency of allele b (three VNTR repeats) of FAM46A gene was significantly increased in the patients compared to the healthy controls in the Croatian and the combined Croatian and Norwegian subjects. Genotype frequencies of cd (four and five VNTR repeats) and cc (four VNTR repeats homozygote) of the FAM46A gene were significantly decreased in the patients compared to the healthy controls in the Croatian and Norwegian subjects, respectively. Logistic regression analyses revealed FAM46A genotype cc to be an independent predictive factor for non small cell lung cancer risk in the Norwegian subjects after adjustment for age, gender and smoking status. This is the first study to suggest an association between the FAM46A gene VNTR polymorphisms and non small cell lung cancer. We found also that BAG6 rs3117582 SNP was associated with non small cell lung cancer in the Norwegian subjects and the combined Croatian-Norwegian subjects corroborating the earlier finding that BAG6 rs3117582 SNP was associated with lung cancer in Europeans. Logistic regression analyses revealed that genotypes and alleles of BAG6 were independent predictive factor for non small cell lung cancer risk in the Norwegian and combined Croatian-Norwegian subjects, after adjustment for age and gender.     

重症急性胰腺炎患者血清IL-6水平与内毒素移位及肠黏膜紧密连接蛋白表达关系的研究

目的：大量研究表明重症急性胰腺炎（SAP）患者血清中高浓度IL-6 和肠黏膜低表达的紧密连接蛋白可促进内毒素移位的发生。本文主要研究重症胰腺炎患者血清IL-6 水平对内毒素移位和肠黏膜紧密连接蛋白表达的影响。方法：50 例重症胰腺炎患者,其中12 例在患病早期因结肠受累合并腹胀,对12 例结肠受累患者应用结肠镜行结肠灌洗进行腹腔减压,同时取结肠黏膜进行活组织检查。所有病人在治疗的第3 天,第7天,第10 天,第14 天抽取外周静脉血。40 例健康志愿者作为对照组。应用ELISA方法检测血清IL-6 水平,鲎试验（LAL）方法检测血清内毒素含量,应用免疫荧光和Western blotting 方法检测肠黏膜紧密连接蛋白表达水平。结果：SAP 患者血清IL-6 和内毒素含量明显高于健康对照组,而结肠黏膜紧密连接蛋白表达低于对照组;在临床治疗过程中,早期SAP 患者血清IL-6 和内毒素水平高于晚期（P 值均＜0.05）。SAP 早期血清高浓度的IL-6 与结肠黏膜紧密连接蛋白的低表达具有相关性,差异有统计学意义（r=0.735,P＜0.05）。结论：血清IL-6 水平可作为早期评价重症急性胰腺炎严重程度的一项指标,IL-6 水平与重症急性胰腺炎临床病程有相关性,可能导致肠道内毒素移位。     

Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins

We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites.     

Expression of SGTA correlates with neuronal apoptosis and reactive gliosis after spinal cord injury

Small glutamine-rich tetratricopeptide repeat (TPR)-containing protein alpha (SGTA) is a novel TPR-containing protein involved in various biological processes. However, the expression and roles of SGTA in the central nervous system remain unknown. We have produced an acute spinal cord injury (SCI) model in adult rats and found that SGTA protein levels first significantly increase, reach a peak at day 3 and then gradually return to normal level at day 14 after SCI. These changes are striking in neurons, astrocytes and microglia. Additionally, colocalization of SGTA/active caspase-3 has been detected in neurons and colocalization of SGTA/proliferating cell nuclear antigen has been detected in astrocytes and microglial. In vitro, SGTA depletion by short interfering RNA inhibits astrocyte proliferation and decreases cyclinA and cyclinD1 protein levels. SGTA knockdown also reduces neuronal apoptosis. We speculate that SGTA is involved in biochemical and physiological responses after SCI.     

重症急性胰腺炎患者血清IL-6水平与内毒素移位及肠黏膜紧密连接蛋白表达关系的研究(英文)

目的:大量研究表明重症急性胰腺炎(SAP)患者血清中高浓度IL-6和肠黏膜低表达的紧密连接蛋白可促进内毒素移位的发生。本文主要研究重症胰腺炎患者血清IL-6水平对内毒素移位和肠黏膜紧密连接蛋白表达的影响。方法:50例重症胰腺炎患者,其中12例在患病早期因结肠受累合并腹胀,对12例结肠受累患者应用结肠镜行结肠灌洗进行腹腔减压,同时取结肠黏膜进行活组织检查。所有病人在治疗的第3天,第7天,第10天,第14天抽取外周静脉血。40例健康志愿者作为对照组。应用ELISA方法检测血清IL-6水平,鲎试验(LAL)方法检测血清内毒素含量,应用免疫荧光和Western blotting方法检测肠黏膜紧密连接蛋白表达水平。结果:SAP患者血清IL-6和内毒素含量明显高于健康对照组,而结肠黏膜紧密连接蛋白表达低于对照组;在临床治疗过程中,早期SAP患者血清IL-6和内毒素水平高于晚期(P值均0.05)。SAP早期血清高浓度的IL-6与结肠黏膜紧密连接蛋白的低表达具有相关性,差异有统计学意义(r=0.735,P0.05)。结论:血清IL-6水平可作为早期评价重症急性胰腺炎严重程度的一项指标,IL-6水平与重症急性胰腺炎临床病程有相关性,可能导致肠道内毒素移位。     

The Expression of Iron-repressible Outer Membrane Proteins in Helicobacter pylori and Its Association with Iron Deficiency Anemia

Background: Helicobacter pylori infection is known to be a cause of iron deficiency anemia (IDA) that is unresponsive to iron supplements. H. pylori bind iron to a specific receptor by iron-repressible outer membrane proteins (IROMPs) under conditions of restricted iron.
Materials and Methods: We compared the expression of IROMPs from strains of H. pylori under both iron-restricted and iron-supplemented conditions to determine the difference between strains with and without IDA. One standard strain, two clinical strains, and three IDA strains were cultured; and then the IROMPs were extracted under iron-restricted and iron-supplemented conditions. We used SDS-PAGE to compare the expression of the IROMPs from each strain.
Results:  IROMPs were found in IDA strains under iron-restricted conditions and their molecular sizes were estimated to be 56, 48, 41, and 37 kDa. In the iron-repleted media, the IROMPs were no longer present.
Conclusion: In the iron-depleted state, specific H. pylori strains associated with IDA demonstrated an advantage in iron acquisition due to a higher expression of IROMPs. Our results can explain in part why some patients with H. pylori infection are more prone to develop clinical IDA under restricted iron conditions in the host.     

Ubiquitination and Degradation of CFTR by the E3 Ubiquitin Ligase MARCH2 through Its Association with Adaptor Proteins CAL and STX6

Golgi-localized cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) and syntaxin 6 (STX6) regulate the abundance of mature, post-ER CFTR by forming a CAL/STX6/CFTR complex (CAL complex) that promotes CFTR degradation in lysosomes. However, the molecular mechanism underlying this degradation is unknown. Here we investigated the interaction of a Golgi-localized, membrane-associated RING-CH E3 ubiquitin ligase, MARCH2, with the CAL complex and the consequent binding, ubiquitination, and degradation of mature CFTR. We found that MARCH2 not only co-immunoprecipitated and co-localized with CAL and STX6, but its binding to CAL was also enhanced by STX6, suggesting a synergistic interaction. In vivo ubiquitination assays demonstrated the ubiquitination of CFTR by MARCH2, and overexpression of MARCH2, like that of CAL and STX6, led to a dose-dependent degradation of mature CFTR that was blocked by bafilomycin A1 treatment. A catalytically dead MARCH2 RING mutant was unable to promote CFTR degradation. In addition, MARCH2 had no effect on a CFTR mutant lacking the PDZ motif, suggesting that binding to the PDZ domain of CAL is required for MARCH2-mediated degradation of CFTR. Indeed, silencing of endogenous CAL ablated the effect of MARCH2 on CFTR. Consistent with its Golgi localization, MARCH2 had no effect on ER-localized ΔF508-CFTR. Finally, siRNA-mediated silencing of endogenous MARCH2 in the CF epithelial cell line CFBE-CFTR increased the abundance of mature CFTR. Taken together, these data suggest that the recruitment of the E3 ubiquitin ligase MARCH2 to the CAL complex and subsequent ubiquitination of CFTR are responsible for the CAL-mediated lysosomal degradation of mature CFTR.     

SRP,FtsY, DnaK and YidC Are Required for the Biogenesis of the E. coli Tail-Anchored Membrane Proteins DjlC and Flk

Tail-anchored membrane proteins (TAMPs) are relatively simple membrane proteins characterized by a single transmembrane domain (TMD) at their C-terminus. Consequently, the hydrophobic TMD, which acts as a subcellular targeting signal, emerges from the ribosome only after termination of translation precluding canonical co-translational targeting and membrane insertion. In contrast to the well-studied eukaryotic TAMPs, surprisingly little is known about the cellular components that facilitate the biogenesis of bacterial TAMPs. In this study, we identify DjlC and Flk as bona fide Escherichia coli TAMPs and show that their TMDs are necessary and sufficient for authentic membrane targeting of the fluorescent reporter mNeonGreen. Using strains conditional for the expression of known E. coli membrane targeting and insertion factors, we demonstrate that the signal recognition particle (SRP), its receptor FtsY, the chaperone DnaK and insertase YidC are each required for efficient membrane localization of both TAMPs. A close association between the TMD of DjlC and Flk with both the Ffh subunit of SRP and YidC was confirmed by site-directed in vivo photo-crosslinking. In addition, our data suggest that the hydrophobicity of the TMD correlates with the dependency on SRP for efficient targeting.