Protein 5A Research Articles

Differential Display of Abundantly Expressed Genes of Trichoderma harzianum During Colonization of Tomato-Germinating Seeds and Roots

The identification of Trichoderma genes whose expression is altered during early stages of interaction with developing roots of germinated seeds is an important step toward understanding the rhizosphere competency of Trichoderma spp. The potential of 13 Trichoderma strains to colonize tomato root and promote plant growth has been evaluated. All used strains successfully propagated in spermosphere and continued their growth in rhizoplane simultaneously root enlargement while the strains T6 and T7 were the most abundant in the apical segment of roots. Root colonization in most strains associated with promoting the roots and shoots growth while they significantly increased up to 43 and 40% roots and shoots dry weights, respectively. Differential display reverse transcriptase-PCR (DDRT-PCR) has been developed to detect differentially expressed genes in the previously selected strain, Trichoderma harzianum T7, during colonization stages of tomato-germinating seeds and roots. Amplified DDRT-PCR products were analyzed on gel agarose and 62 differential bands excised, purified, cloned, and sequenced. Obtained ESTs were submit-queried to NCBI database by BLASTx search and gene ontology hierarchy. Most of transcripts (29 EST) corresponds to known and hypothetical proteins such as secretion-related small GTPase, 40S ribosomal protein S3a, 3-hydroxybutyryl-CoA dehydrogenase, DNA repair protein rad50, lipid phosphate phosphatase-related protein type 3, nuclear essential protein, phospholipase A2, fatty acid desaturase, nuclear pore complex subunit Nup133, ubiquitin-activating enzyme, and 60S ribosomal protein L40. Also, 13 of these sequences showed no homology (E>0.05) with public databases and considered as novel genes. Some of these ESTs corresponded to genes encodes enzymes potentially involved in nutritional support of microorganisms which have obvious importance in the establishment of Trichoderma in spermosphere and rhizosphere, via potentially functioning in acquisition of nutrients from energy-rich carbon compounds leaked from the germinating seeds and roots.

The promiscuous binding of the Fyn SH3 domain to a peptide from the NS5A protein

The hepatitis C virus nonstructural 5A (NS5A) protein is a large zinc-binding phosphoprotein that plays an important role in viral RNA replication and is involved in altering signal transduction pathways in the host cell. This protein interacts with Fyn tyrosine kinase in vivo and regulates its kinase activity. The 1.5 Å resolution crystal structure of a complex between the SH3 domain of the Fyn tyrosine kinase and the C-terminal proline-rich motif of the NS5A-derived peptide APPIPPPRRKR has been solved. Crystals were obtained in the presence of ZnCl(2) and belonged to the tetragonal space group P4(1)2(1)2. The asymmetric unit is composed of four SH3 domains and two NS5A peptide molecules; only three of the domain molecules contain a bound peptide, while the fourth molecule seems to correspond to a free form of the domain. Additionally, two of the SH3 domains are bound to the same peptide chain and form a ternary complex. The proline-rich motif present in the NS5A protein seems to be important for RNA replication and virus assembly, and the promiscuous interaction of the Fyn SH3 domain with the NS5A C-terminal proline-rich peptide found in this crystallographic structure may be important in the virus infection cycle.

A Member of a New Picornaviridae Genus Is Shed in Pig Feces

During a study of the fecal microbiomes from two healthy piglets using high-throughput sequencing (HTS), we identified a viral genome containing an open reading frame encoding a predicted polyprotein of 2,133 amino acids. This novel viral genome displayed the typical organization of picornaviruses, containing three structural proteins (VP0, VP3, and VP1), followed by seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C(pro), and 3D(pol)). Given its particular relationship with Parechovirus, we propose to name it "Pasivirus" for Parecho sister clade virus, with "Swine pasivirus 1" (SPaV1) as the type species. Fecal samples collected at an industrial farm from healthy sows and piglets from the same herd (25 and 75, respectively) with ages ranging from 4 to 28 weeks were analyzed for the presence of SPaV1 by one-step reverse transcription (RT)-PCR targeting a 3D region of 151 bp. SPaV1 was detected in fecal samples from 51/75 healthy piglets (68% of the animals) and in none of the 25 fecal samples from healthy sows, indicating that SPaV1 circulates through enteric infection of healthy piglets. We propose that SPaV1 represents the first member of a novel Picornaviridae genus related to parechoviruses.

The Cyclophilin Inhibitor SCY-635 Disrupts Hepatitis C Virus NS5A-Cyclophilin A Complexes

The nonimmunosuppressive cyclophilin (Cyp) inhibitor SCY-635 blocks hepatitis C virus (HCV) replication both in vitro and in vivo and represents a novel potent anti-HCV agent. However, its mechanism of action remains to be fully elucidated. A growing body of evidence suggests that cyclophilin A (CypA) is absolutely necessary for HCV replication and that the HCV nonstructural 5A (NS5A) protein serves as a main viral ligand for CypA. In this study, we examined the effect of SCY-635 on HCV replication. Specifically, we asked whether SCY-635 blocks HCV replication by targeting CypA-NS5A interactions. We also investigated the possibility that HCV can escape SCY-635 selection pressure and whether this resistance influences either CypA-NS5A interactions or the dependence of HCV on CypA. We found not only that SCY-635 efficiently inhibits HCV replication, but it is sufficient alone to clear HCV replicon-containing cells. We found that SCY-635 prevents CypA-NS5A interactions in a dose-dependent manner. SCY-635 prevents the contact between CypA and NS5A derived from genotypes 1 to 3. Together, these data suggest that NS5A-CypA interactions control HCV replication and that SCY-635 blocks viral replication by preventing the formation of these complexes. We also found that NS5A mutant proteins found in SCY-635-resistant HCV replicons behave similarly to wild-type NS5A in terms of both CypA binding and SCY-635-mediated dissociation and inhibition of CypA binding. However, the NS5A mutations found in SCY-635-resistant HCV replicons rescued viral replication in CypA-knockdown cells, suggesting that the NS5A mutations, which arose in vitro under SCY-635 selection, do not alter the binding affinity of CypA for NS5A. These specific mutations in NS5A eliminate the dependence of HCV RNA replication on the expression of host CypA.

Mutations in AEC syndrome skin reveal a role for p63 in basement membrane adhesion, skin barrier integrity and hair follicle biology

AEC (ankyloblepharon-ectodermal defects-clefting) syndrome is an autosomal dominant ectodermal dysplasia disorder caused by mutations in the transcription factor p63. Clinically, the skin is dry and often fragile; other features can include partial eyelid fusion (ankyloblepharon), hypodontia, orofacial clefting, sparse hair or alopecia, and nail dystrophy. To investigate how p63 gene mutations affect gene and protein expression in AEC syndrome skin. We performed microarray analysis on samples of intact and eroded AEC syndrome skin compared with control skin. Changes were verified by quantitative real-time reverse transcription-polymerase chain reaction and, for basal keratinocyte-associated genes, by immunohistochemistry and analysis of microdissected skin. We identified significant upregulation of six genes and downregulation of 69 genes in AEC syndrome skin, with the main changes in genes implicated in epidermal adhesion, skin barrier formation and hair follicle biology. There was reduced expression of genes encoding the basement membrane proteins FRAS1 and collagen VII, as well as the skin barrier-associated small proline-rich proteins 1A and 4, late cornified envelope protein 5A, hornerin, and lipid transporters including ALOX15B. Reduced expression of the hair-associated keratins 25, 27, 31, 33B, 34, 35, 81 and 85 was also noted. We also confirmed similar alterations in gene expression for 26 of the 75 genes in eroded AEC scalp skin. This study identifies specific changes in skin structural biology and signalling pathways that result from mutant p63 and provides new molecular insight into the AEC syndrome phenotype.

Loss of Cytoskeletal Transport during Egress Critically Attenuates Ectromelia Virus Infection In Vivo

Egress of wrapped virus (WV) to the cell periphery following vaccinia virus (VACV) replication is dependent on interactions with the microtubule motor complex kinesin-1 and is mediated by the viral envelope protein A36. Here we report that ectromelia virus (ECTV), a related orthopoxvirus and the causative agent of mousepox, encodes an A36 homologue (ECTV-Mos-142) that is highly conserved despite a large truncation at the C terminus. Deleting the ECTV A36R gene leads to a reduction in the number of extracellular viruses formed and to a reduced plaque size, consistent with a role in microtubule transport. We also observed a complete loss of virus-associated actin comets, another phenotype dependent on A36 expression during VACV infection. ECTV ΔA36R was severely attenuated when used to infect the normally susceptible BALB/c mouse strain. ECTV ΔA36R replication and spread from the draining lymph nodes to the liver and spleen were significantly reduced in BALB/c mice and in Rag-1-deficient mice, which lack T and B lymphocytes. The dramatic reduction in ECTV ΔA36R titers early during the course of infection was not associated with an augmented immune response. Taken together, these findings demonstrate the critical role that subcellular transport pathways play not only in orthopoxvirus infection in an in vitro context but also during orthopoxvirus pathogenesis in a natural host. Furthermore, despite the attenuation of the mutant virus, we found that infection nonetheless induced protective immunity in mice, suggesting that orthopoxvirus vectors with A36 deletions may be considered another safe vaccine alternative.

A cell permeable hairpin peptide inhibits hepatitis C viral NS5A‐mediated translation and virus production

Non‐structural protein 5A (NS5A) is a key regulator of the hepatitis C virus (HCV) life cycle including RNA replication, assembly and HCV internal ribosome entry site (IRES)‐mediated translation. We have previously identified an NS5A/heat shock protein 70 (HSP70) complex that is important for HCV IRES mediated translation. We have mapped the HSP70 interaction site on NS5A to a beta‐sheet hairpin in domain 1. Mutation of this site reduced NS5A‐augmented IRES‐mediated translation. We designed a cell permeable hairpin peptide derivative of this site which blocked NS5A/HSP70 complex formation in vitro compared to scrambled control. This peptide also inhibited IRES‐mediated translation, viral RNA replication and infectious virion production in a dose‐dependent manner. These results support the hypothesis that the NS5A/HSP70 complex is involved in IRES‐mediated translation and identify a sequence specific element in NS5A responsible for complex formation. Identification of this element will allow for further interrogation of NS5A‐mediated IRES activity, sequence specific HSP recognition and rational drug design.

Methylation of translation‐associated proteins in Saccharomyces cerevisiae: Identification of methylated lysines and their methyltransferases

This study aimed to identify sites of lysine methylation in Saccharomyces cerevisiae and the associated methyltransferases. Hexapeptide ligand affinity chromatography was used to normalize the abundance levels of proteins in whole cell lysate. MS/MS, in association with antibody-based detection, was then used to identify lysine methylated proteins and the precise sites of modification. Lysine methylation was found on the proteins elongation factor (EF) 1-α, 2, and 3A, as well as ribosomal proteins 40S S18-A/B, 60S L11-A/B, L18-A/B, and L42-A/B. Precise sites were mapped in all cases. Single-gene knockouts of known and putative methyltransferase(s), in association with MS/MS, showed that EF1-α is monomethylated by Efm1 at lysin 30 and dimethylated by See1 at lysine 316. Methyltransferase Rkm1 was found to monomethylate 40S ribosomal protein S18-A/B at lysine 48. Knockout analysis also revealed that putative methyltransferase YBR271W affects the methylation of proteins EF2 and 3A; this was detected by Western blotting and immunodetection. This methyltransferase shows strong interspecies conservation and a tryptophan-containing motif associated with its active site. We suggest that enzyme YBR271W is named EF methyltransferase 2 (Efm2), in line with the recent naming of YHL039W as Efm1.

3T3 Cell Lines Stably Expressing Pax6 or Pax6(5a) – A New Tool Used for Identification of Common and Isoform Specific Target Genes

Pax6 and Pax6(5a) are two isoforms of the evolutionary conserved Pax6 gene often co-expressed in specific stochiometric relationship in the brain and the eye during development. The Pax6(5a) protein differs from Pax6 by having a 14 amino acid insert in the paired domain, causing the two proteins to have different DNA binding specificities. Difference in functions during development is proven by the fact that mutations in the 14 amino acid insertion for Pax6(5a) give a slightly different eye phenotype than the one described for Pax6. Whereas quite many Pax6 target genes have been published during the last years, few Pax6(5a) specific target genes have been reported on. However, target genes identified by Pax6 knockout studies can probably be Pax6(5a) targets as well, since this isoform also will be affected by the knockout. In order to identify new Pax6 target genes, and to try to distinguish between genes regulated by Pax6 and Pax6(5a), we generated FlpIn-3T3 cell lines stably expressing Pax6 or Pax6(5a). RNA was harvested from these cell lines and used in gene expression microarrays where we identified a number of genes differentially regulated by Pax6 and Pax6(5a). A majority of these were associated with the extracellular region. By qPCR we verified that Ncam1, Ngef, Sphk1, Dkk3 and Crtap are Pax6(5a) specific target genes, while Tgfbi, Vegfa, EphB2, Klk8 and Edn1 were confirmed as Pax6 specific target genes. Nbl1, Ngfb and seven genes encoding different glycosyl transferases appeared to be regulated by both. Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP. Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities.

A DNA vaccination regime including protein boost and electroporation protects cattle against foot-and-mouth disease

Protection against foot-and-mouth disease (FMD) using DNA technology has been documented for sheep and pigs but not for the highly susceptible species of cattle.Twenty-five Holstein Friesian cross-bred cattle were vaccinated twice, 21days apart, with a DNA vaccine containing the capsid coding region (P1) along with the non-structural proteins 2A, 3C and 3D (pcDNA3.1/P1-2A3C3D) of O1 Kaufbeuren alone or coated onto PLG (d,l-lactide-co-glycolide) microparticles. In some pcDNA3.1/P1-2A3C3D was also combined with an adjuvant plasmid expressing bovine granulocyte macrophage colony stimulating factor (GM-CSF). DNA vaccinations were administered intramuscularly with, or without, the use of electroporation and at 42days post primary vaccination cattle received a protein boost of 146S FMD virus (FMDV) antigen and non-structural protein 3D. For comparison, four cattle were vaccinated with a conventional FMD vaccine and two more included as unvaccinated controls. Apart from those immunised with PLG microparticles all cattle were challenged with 105 TCID50 cattle adapted O1 Lausanne FMDV virus at day 93 post primary vaccination.All DNA vaccinated cattle regardless of regime developed good humoral and cell mediated responses prior to challenge. The best overall virus neutralising antibody, IFN-γ and clinical protection (75%) were seen in the cattle whereby the DNA was delivered by electroporation. In contrast, only 25% of cattle vaccinated with the DNA vaccine without electroporation were clinically protected. The addition of GM-CSF in combination with electroporation further improved the efficacy of the vaccine, as demonstrated from the reduction of clinical disease and virus excretions in nasal swabs.We thus demonstrate for the first time that cattle can be clinically protected against FMDV challenge following a DNA prime-protein boost strategy, and particularly when DNA vaccine is combined with GM-CSF and delivered by electroporation.

Model membrane platforms for biomedicine: case study on antiviral drug development.

As one of the most important interfaces in cellular systems, biological membranes have essential functions in many activities such as cellular protection and signaling. Beyond their direct functions, they also serve as scaffolds to support the association of proteins involved in structural support, adhesion, and transport. Unfortunately, biological processes sometimes malfunction and require therapeutic intervention. For those processes which occur within or upon membranes, it is oftentimes difficult to study the mechanism in a biologically relevant, membranous environment. Therefore, the identification of direct therapeutic targets is challenging. In order to overcome this barrier, engineering strategies offer a new approach to interrogate biological activities at membrane interfaces by analyzing them through the principles of the interfacial sciences. Since membranes are complex biological interfaces, the development of simplified model systems which mimic important properties of membranes can enable fundamental characterization of interaction parameters for such processes. We have selected the hepatitis C virus (HCV) as a model viral pathogen to demonstrate how model membrane platforms can aid antiviral drug discovery and development. Responsible for generating the genomic diversity that makes treating HCV infection so difficult, viral replication represents an ideal step in the virus life cycle for therapeutic intervention. To target HCV genome replication, the interaction of viral proteins with model membrane platforms has served as a useful strategy for target identification and characterization. In this review article, we demonstrate how engineering approaches have led to the discovery of a new functional activity encoded within the HCV nonstructural 5A protein. Specifically, its N-terminal amphipathic, α-helix (AH) can rupture lipid vesicles in a size-dependent manner. While this activity has a number of exciting biotechnology and biomedical applications, arguably the most promising one is in antiviral medicine. Based on the similarities between lipid vesicles and the lipid envelopes of virus particles, experimental findings from model membrane platforms led to the prediction that a range of medically important viruses might be susceptible to rupturing treatment with synthetic AH peptide. This hypothesis was tested and validated by molecular virology studies. Broad-spectrum antiviral activity of the AH peptide has been identified against HCV, HIV, herpes simplex virus, and dengue virus, and many more deadly pathogens. As a result, the AH peptide is the first in class of broad-spectrum, lipid envelope-rupturing antiviral agents, and has entered the drug pipeline. In summary, engineering strategies break down complex biological systems into simplified biomimetic models that recapitulate the most important parameters. This approach is particularly advantageous for membrane-associated biological processes because model membrane platforms provide more direct characterization of target interactions than is possible with other methods. Consequently, model membrane platforms hold great promise for solving important biomedical problems and speeding up the translation of biological knowledge into clinical applications.

Expression of the hepatic skatole- and androstenone-metabolising enzymes in entire male pigs of two live weights

The aim of the present study was to investigate an in vivo relationship between increased levels of testicular steroids and protein expression of the key enzymes involved in hepatic metabolism of skatole (cytochrome P4502E1 and CYP2E1 and CYP2A) and androstenone (3β-hydroxysteroid dehydrogenase — 3β-HSD, and sulfotransferase SULT2B1). For this purpose, we compared the protein expression in the liver from high weight (115kg) and low weight (90kg) pigs and related it to the level of the testicular steroids testosterone, oestrone sulphate and androstenone. We demonstrated that protein expressions of both skatole and androstenone-metabolising enzymes were affected by live weight of pigs. Expressions of CYP2E1 and 2A proteins were significantly lower (P=0.05 and 0.001, respectively) in the liver microsomes of high weight pigs compared to that of low weight pigs. The expression of androstenone-metabolising 3β-HSD was also lower (P=0.003) in high weight pigs whilst the expression of SULT2B1 was higher in high weight pigs (P=0.036). We concluded that the age-related decrease in activities of cytochrome P4502E1 and 2A and thus increased fat skatole levels are likely due to decreased protein expression of those enzymes in older (heavier) pigs with higher levels of testicular steroids. The age-related increase in androstenone levels can be at least partly due to decreased protein expression of 3β-hydroxysteroid dehydrogenase, but not SULT2B1.

Deletion of penicillin-binding protein 1b impairs biofilm formation and motility in Escherichia coli

The major bifunctional transpeptidases/transglycosylases of Escherichia coli, penicillin-binding proteins (PBPs) 1a and 1b, were evaluated for their influence on biofilm formation. While the PBP1a mutant was unaffected, the PBP1b mutant exhibited significantly decreased biofilm formation and motility. Interestingly, the extracellular indole concentration was higher in the PBP1b mutant, and similar phenotypic defects were replicated in the wild-type upon addition of exogenous indole. Expression of PBP1b in trans substantially decreased indole production and restored normal phenotypes. Results further suggest that rpoS deletion has a counteracting effect on the mrcB mutant. These findings indicate that PBP1b deletion influences biofilm formation and motility, possibly through indole.

The roles of ribosomal protein S3a in ovarian development of Fenneropenaeus merguiensis (De Man)

Shrimp ovarian development is a pivotal step for aquaculture in order to maintain broodstocks. The molecular processes in the ovaries of Fenneropenaeus merguiensis De Man during vitellogenesis were investigated. A quantitative real time PCR (Q-PCR) assay showed a high expression of ribosomal protein S3a (RPS3a) in the early stage of ovarian development and a decline in the later stages. Serotonin (5-HT) increased the expression of RPS3a significantly at 4h after incubating with the ovarian explants. A full-length RPS3a was amplified by the RACE technique and expressed in Escherichia coli BL21 (DE3). Western blot analysis showed that the molecular weight of the recombinant histidine (His)-RPS3a was approximately 33kDa. Its function was tested by incubating undeveloped ovarian explants with His-RPS3a. In vitro assays indicated that the His-RPS3a stimulated the expression of shrimp ovarian peritrophin (SOP) and translational controlled tumor protein (TCTP) genes significantly when compared to the untreated ovarian explants. In addition, the SOP and TCTP genes had been previously reported to be highly expressed during the early stages of ovarian development. In addition, FITC-RPS3a was shown to be imported into the ovarian cells. Therefore, RPS3a may act as a stimulator for the development of shrimp ovaries.

Vaccinia Mature Virus Fusion Regulator A26 Protein Binds to A16 and G9 Proteins of the Viral Entry Fusion Complex and Dissociates from Mature Virions at Low pH

Vaccinia mature virus enters cells through either endocytosis or plasma membrane fusion, depending on virus strain and cell type. Our previous results showed that vaccinia virus mature virions containing viral A26 protein enter HeLa cells preferentially through endocytosis, whereas mature virions lacking A26 protein enter through plasma membrane fusion, leading us to propose that A26 acts as an acid-sensitive fusion suppressor for mature virus (S. J. Chang, Y. X. Chang, R. Izmailyan R, Y. L. Tang, and W. Chang, J. Virol. 84:8422-8432, 2010). In the present study, we investigated the fusion suppression mechanism of A26 protein. We found that A26 protein was coimmunoprecipitated with multiple components of the viral entry-fusion complex (EFC) in infected HeLa cells. Transient expression of viral EFC components in HeLa cells revealed that vaccinia virus A26 protein interacted directly with A16 and G9 but not with G3, L5 and H2 proteins of the EFC components. Consistently, a glutathione S-transferase (GST)-A26 fusion protein, but not GST, pulled down A16 and G9 proteins individually in vitro. Together, our results supported the idea that A26 protein binds to A16 and G9 protein at neutral pH contributing to suppression of vaccinia virus-triggered membrane fusion from without. Since vaccinia virus extracellular envelope proteins A56/K2 were recently shown to bind to the A16/G9 subcomplex to suppress virus-induced fusion from within, our results also highlight an evolutionary convergence in which vaccinia viral fusion suppressor proteins regulate membrane fusion by targeting the A16 and G9 components of the viral EFC complex. Finally, we provide evidence that acid (pH 4.7) treatment induced A26 protein and A26-A27 protein complexes of 70 kDa and 90 kDa to dissociate from mature virions, suggesting that the structure of A26 protein is acid sensitive.

New insights regarding HCV-NS5A structure/function and indication of genotypic differences

BackgroundHCV is prevalent throughout the world. It is a major cause of chronic liver disease. There is no effective vaccine and the most common therapy, based on Peginterferon, has a success rate of ~50%. The mechanisms underlying viral resistance have not been elucidated but it has been suggested that both host and virus contribute to therapy outcome. Non-structural 5A (NS5A) protein, a critical virus component, is involved in cellular and viral processes.MethodsThe present study analyzed structural and functional features of 345 sequences of HCV-NS5A genotypes 1 or 3, using in silico tools.ResultsThere was residue type composition and secondary structure differences between the genotypes. In addition, second structural variance were statistical different for each response group in genotype 3. A motif search indicated conserved glycosylation, phosphorylation and myristoylation sites that could be important in structural stabilization and function. Furthermore, a highly conserved integrin ligation site was identified, and could be linked to nuclear forms of NS5A. ProtFun indicated NS5A to have diverse enzymatic and nonenzymatic activities, participating in a great range of cell functions, with statistical difference between genotypes.ConclusionThis study presents new insights into the HCV-NS5A. It is the first study that using bioinformatics tools, suggests differences between genotypes and response to therapy that can be related to NS5A protein features. Therefore, it emphasizes the importance of using bioinformatics tools in viral studies. Data acquired herein will aid in clarifying the structure/function of this protein and in the development of antiviral agents.

Hepatic expression and cellular distribution of the glucose transporter family

Glucose and other carbohydrates are transported into cells using members of a family of integral membrane glucose transporter (GLUT) molecules. To date 14 members of this family, also called the solute carrier 2A proteins have been identified which are divided on the basis of transport characteristics and sequence similarities into several families (Classes 1 to 3). The expression of these different receptor subtypes varies between different species, tissues and cellular subtypes and each has differential sensitivities to stimuli such as insulin. The liver is a contributor to metabolic carbohydrate homeostasis and is a major site for synthesis, storage and redistribution of carbohydrates. Situations in which the balance of glucose homeostasis is upset such as diabetes or the metabolic syndrome can lead metabolic disturbances that drive chronic organ damage and failure, confirming the importance of understanding the molecular regulation of hepatic glucose homeostasis. There is a considerable literature describing the expression and function of receptors that regulate glucose uptake and release by hepatocytes, the most import cells in glucose regulation and glycogen storage. However there is less appreciation of the roles of GLUTs expressed by non parenchymal cell types within the liver, all of which require carbohydrate to function. A better understanding of the detailed cellular distribution of GLUTs in human liver tissue may shed light on mechanisms underlying disease pathogenesis. This review summarises the available literature on hepatocellular expression of GLUTs in health and disease and highlights areas where further investigation is required.

3CD, but not 3C, cleaves the VP1/2A site efficiently during Aichi virus polyprotein processing through interaction with 2A

Picornavirus genomes are translated into a single large polyprotein, which is processed by virus-encoded proteases into individual functional proteins. 3C of all picornaviruses is a protease, and the leader (L) and 2A proteins of some picornaviruses are also involved in polyprotein processing. Aichi virus (AiV), which is associated with acute gastroenteritis in humans, is a member of the genus Kobuvirus of the family Picornaviridae. The AiV L and 2A proteins have already been shown to exhibit no protease activity. In this study, we investigated AiV polyprotein processing by 3C and 3CD using a cell-free translation system. 3C and 3CD were capable of processing the polyprotein in trans; 3C, however, cleaved the VP1/2A site inefficiently, while 3CD cleaved this site almost completely. Mammalian two-hybrid and coimmunoprecipitation assays showed an interaction between 2A and 3CD. Using a 3CD mutant and various 2A mutants of substrate proteins, we showed a clear correlation between the 2A–3CD interaction and the VP1/2A cleavage by 3CD. Thus, this study suggests that tight interaction of 3CD with the 2A region of a precursor protein is required for efficient cleavage at the VP1/2A site.

A20 and ABIN-3 possibly promote regression of trehalose 6,6′-dimycolate (TDM)-induced granuloma by interacting with an NF-kappa B signaling protein, TAK-1

The objective of this paper is to examine the role of NF-kappa B inhibitors A20 and ABIN-family proteins in the trehalose 6,6'-dimycolate (TDM)-induced model of tuberculous granulomatous lesions. BALB/c mice were twice injected i.p. with w/o/w emulsions that contain TDM at a 1week-interval. The mice were killed at days 0, 3, 7, 14, or 21 after the last injection. The mRNA and protein levels of A20 and ABIN-family proteins were measured by real-time PCR using mRNA or protein extract from the lesions. The activation status of NF-kappa B was analyzed by Western blotting and immunohistochemistry. Finally, the protein extracts were immunoprecipitated by anti-ABIN-3 antibody to identify the protein that potentially interacts with ABIN-3. The activation of NF-kappa B pathway coincided with granuloma development, while A20 and ABIN-3 increased in accordance with granuloma regression. TAK-1 protein was co-precipitated with ABIN-3 by immunoprecipitation using anti-ABIN-3 antibody. The results suggest that ABIN-3 contributed to granuloma regression by interacting with TAK-1 and, as a consequence, inhibiting activation of NF-kappa B pathway.

A novel class of PTEN protein in Arabidopsis displays unusual phosphoinositide phosphatase activity and efficiently binds phosphatidic acid

PTEN (phosphatase and tensin homologue deleted on chromosome ten) proteins are dual phosphatases with both protein and phosphoinositide phosphatase activity. They modulate signalling pathways controlling growth, metabolism and apoptosis in animals and are implied in several human diseases. In the present paper we describe a novel class of PTEN pro-teins in plants, termed PTEN2, which comprises the AtPTEN (Arabidopsis PTEN) 2a and AtPTEN2b proteins in Arabidopsis. Both display low in vitro tyrosine phosphatase activity. In addition, AtPTEN2a actively dephosphorylates in vitro the 3' phosphate group of PI3P (phosphatidylinositol 3-phosphate), PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate) and PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate). In contrast with animal PTENs, PI(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate) is a poor substrate. Site-directed mutagenesis of AtPTEN2a and molecular modelling of protein-phosphoinositide interactions indicated that substitutions at the PTEN2 core catalytic site of the Lys267 and Gly268 residues found in animals, which are critical for animal PTEN activity, by Met267 and Ala268 found in the eudicot PTEN2 are responsible for changes in substrate specificity. Remarkably, the AtPTEN2a protein also displays strong binding activity for PA (phosphatidic acid), a major lipid second messenger in plants. Promoter::GUS (β-glucuronidase) fusion, transcript and protein analyses further showed the transcriptional regulation of the ubiquitously expressed AtPTEN2a and AtPTEN2b by salt and osmotic stress. The results of the present study suggest a function for this novel class of plant PTEN proteins as an effector of lipid signalling in plants.