Nrbf2 Protein Suppresses Autophagy by Modulating Atg14L Protein-containing Beclin 1-Vps34 Complex Architecture and Reducing Intracellular Phosphatidylinositol-3 Phosphate Levels

Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1^−/−;Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy.     

A Phase I Trial of DFMO Targeting Polyamine Addiction in Patients with Relapsed/Refractory Neuroblastoma

BackgroundNeuroblastoma (NB) is the most common cancer in infancy and most frequent cause of death from extracranial solid tumors in children. Ornithine decarboxylase (ODC) expression is an independent indicator of poor prognosis in NB patients. This study investigated safety, response, pharmacokinetics, genetic and metabolic factors associated with ODC in a clinical trial of the ODC inhibitor difluoromethylornithine (DFMO) ± etoposide for patients with relapsed or refractory NB.ConclusionsDFMO doses of 500-1500mg/m2/day are safe and well tolerated in children with relapsed NB. Children with the minor T allele at rs2302616 of the ODC gene with relapsed or refractory NB had higher levels of urinary polyamine markers and responded better to therapy containing DFMO, compared to those with the major G allele at this locus. These findings suggest that this patient subset may display dependence on polyamines and be uniquely susceptible to therapies targeting this pathway.

Trial Registration

Clinicaltrials.gov NCT#01059071     

A Nonsense Mutation in a Cinnamyl Alcohol Dehydrogenase Gene Is Responsible for the Sorghum brown midrib6 Phenotype

brown midrib6 (bmr6) affects phenylpropanoid metabolism, resulting in reduced lignin concentrations and altered lignin composition in sorghum (Sorghum bicolor). Recently, bmr6 plants were shown to have limited cinnamyl alcohol dehydrogenase activity (CAD; EC 1.1.1.195), the enzyme that catalyzes the conversion of hydroxycinnamoyl aldehydes (monolignals) to monolignols. A candidate gene approach was taken to identify Bmr6. Two CAD genes (Sb02g024190 and Sb04g005950) were identified in the sorghum genome based on similarity to known CAD genes and through DNA sequencing a nonsense mutation was discovered in Sb04g005950 that results in a truncated protein lacking the NADPH-binding and C-terminal catalytic domains. Immunoblotting confirmed that the Bmr6 protein was absent in protein extracts from bmr6 plants. Phylogenetic analysis indicated that Bmr6 is a member of an evolutionarily conserved group of CAD proteins, which function in lignin biosynthesis. In addition, Bmr6 is distinct from the other CAD-like proteins in sorghum, including SbCAD4 (Sb02g024190). Although both Bmr6 and SbCAD4 are expressed in sorghum internodes, an examination of enzymatic activity of recombinant Bmr6 and SbCAD4 showed that Bmr6 had 1 to 2 orders of magnitude greater activity for monolignol substrates. Modeling of Bmr6 and SbCAD4 protein structures showed differences in the amino acid composition of the active site that could explain the difference in enzyme activity. These differences include His-57, which is unique to Bmr6 and other grass CADs. In summary, Bmr6 encodes the major CAD protein involved in lignin synthesis in sorghum, and the bmr6 mutant is a null allele.Plant cell walls constitute a vast reserve of fixed carbon. Cellulose and lignin are the first and second most abundant polymers on the planet, respectively (Jung and Ni, 1998). The world community has started to look to biomass as substrates for plant-based biologically sustainable fuels, which would mitigate carbon dioxide emission and reduce petroleum dependence (Sarath et al., 2008; Schmer et al., 2008). In the current generation of biofuels, ethanol is being synthesized via the fermentation of grain starch or sugarcane juice. For the next generation of biofuels, research is being directed toward the conversion of lignocellulosic biomass into biofuels (Chang, 2007). As bioenergy technologies progress, the conversion of biomass to biofuels could involve a range of chemical, biochemical, and fermentation processes to produce biofuels; alternate biofuels, such as butanol or dimethylfuran, are also on the horizon (Ezeji et al., 2007; Roman-Leshkov et al., 2007). Most liquid biofuel production processes will likely rely on the conversion of the cell wall polysaccharides cellulose and hemicellulose into monomeric sugars.Plant cell walls consist of a complex polysaccharide moiety composed of cellulose microfibrils, composed of β-1,4-linked Glc polymers (Carpita and McCann, 2000). Connecting the cellulose microfibrils to each other is a hemicellulose network, whose structure and composition are species dependent, and which is mainly composed of glucuronoarabinoxylans in grasses (Carpita and McCann, 2000). Lignin, a nonlinear heterogeneous polymer derived from aromatic precursors, cross-links these polysaccharides, rigidifying and reinforcing the cell wall structure (Carpita and McCann, 2000). The addition of lignin polymers to the polysaccharide matrix creates a barrier that is chemically and microbially resistant.Lignin can block the liberation of sugars from the cell wall polysaccharide moieties, release compounds that can inhibit microbes used for fermenting sugars to fuels, and adhere to hydrolytic enzymes. Understanding lignin synthesis, structure, and function to increase cell wall digestibility has long been a goal for forage improvement and paper processing (Mackay et al., 1997; Jung and Ni, 1998). Recently, manipulating lignin has also become an important target for bioenergy feedstock improvement (Chen and Dixon, 2007; Li et al., 2008).Lignin is derived from the phenylpropanoid pathway and contains primarily three types of phenolic subunits: p-hydroxyphenyl, guaiacyl, and syringyl units (Dixon et al., 2001). The phenolic aldehyde precursors are reduced into their corresponding alcohols (monolignols) and subsequently transported to the cell wall (Fig. 1), where laccases and peroxidases catalyze lignin polymerization through the formation of monolignol radicals (Boerjan et al., 2003). Therefore, most research efforts to manipulate lignin have focused on biosynthesis of the monolignols. Most of the enzymes involved in monolignol synthesis have been cloned and characterized in Arabidopsis (Arabidopsis thaliana) and other dicot species, using both mutagenic and transgenic approaches to study the impact of these gene products on dicot cell walls (Anterola and Lewis, 2002). However, there are significant differences in the architecture, polysaccharide composition, and phenylpropanoid composition of grass cell walls compared with those of dicots (Carpita and McCann, 2000; Vogel and Jung, 2001). For example, grasses contain significant amounts of p-coumaric acid and ferulic acid that are cross-linked to cell wall polysaccharides through ester and ether linkages in addition to their presence in lignin (Grabber et al., 1991; Boerjan et al., 2003). Because many of the proposed dedicated bioenergy crops are grasses, there is a need to identify and understand the function of the gene products involved in lignin biosynthesis in these species (Vermerris et al., 2007; Li et al., 2008; Sarath et al., 2008).Open in a separate windowFigure 1.The CAD enzyme and its role in the monolignol biosynthetic pathway. A, CAD catalyzes the conversion of cinnamyl aldehydes to alcohols using NADPH as its cofactor. p-Coumaryl aldehyde and alcohol, R₁ and R₂ = H; caffeoyl aldehyde and alcohol, R₁ and R₂ = OH; coniferyl aldehyde and alcohol, R₁ = H and R₂ = OCH₃; sinapyl aldehyde and alcohol, R₁ and R₂ = OCH₃. B, A simplified model of the lignin biosynthetic pathway where CAD catalyzes the final step in monolignol biosynthesis.The brown midrib phenotype has been useful for identifying mutants affecting lignin synthesis in grasses because it is a visible phenotype. Spontaneous brown midrib mutants were first discovered in maize (Zea mays; Jorgenson, 1931) and were subsequently generated in sorghum (Sorghum bicolor) using diethyl sulfate mutagenesis (Porter et al., 1978). Brown midrib mutants in maize, sorghum, and pearl millet (Pennisetum glaucum) have increased forage digestibility for livestock (Cherney et al., 1990; Akin et al., 1993; Jung et al., 1998; Oliver et al., 2004). In maize and sorghum, there are at least four brown midrib loci in their respective genomes (Jorgenson, 1931; Porter et al., 1978; Gupta, 1995). The genes encoding bm3 in maize and bmr12 in sorghum are the only loci cloned to date, and both encode highly similar caffeic acid O-methyl transferases (Vignols et al., 1995; Bout and Vermerris, 2003). A second brown midrib locus associated with reduced cinnamyl alcohol dehydrogenase (CAD) activity has been identified both in maize (bm1; Halpin et al., 1998) and sorghum (bmr6; Bucholtz et al., 1980; Pillonel et al., 1991). CAD is a member of the alcohol dehydrogenase superfamily of proteins that catalyzes the conversion of the hydroxycinnamoyl aldehydes into alcohols prior to their incorporation into lignin polymers (Fig. 1). Reduced CAD activity results in increased digestibility on dry weight basis, altered cell wall architecture, reduced lignin level, and the incorporation of phenolic aldehydes into lignin in sorghum and maize (Pillonel et al., 1991; Provan et al., 1997; Halpin et al., 1998; Marita et al., 2003; Shi et al., 2006; Palmer et al., 2008). The reduced CAD activity in bm1 has been genetically mapped to a region of the maize genome that contained a CAD gene, ZmCAD2 (Halpin et al., 1998), but a mutation was not identified. However, it has recently been shown that bm1 down-regulated the expression of several lignin biosynthetic genes, suggesting its gene product may be a regulatory protein (Shi et al., 2006; Guillaumie et al., 2007).To identify the mutation responsible for the bmr6 phenotype and to characterize how bmr6 impacts the lignin biosynthetic pathway, a candidate gene approach was taken. Here, we describe the cloning and characterization of Bmr6 and a related protein, SbCAD4. The identification and characterization of Bmr6 has revealed the major monolignol CAD protein in the grasses, which is likely to aid the development of new strategies to increase conversion of sorghum and other grass feedstocks to biofuels.     

Bacterial,archaeal and eukaryal community structures throughout soil horizons of harvested and naturally disturbed forest stands

Disturbances caused by timber harvesting have critical long‐term effects on the forest soil microbiota and alter fundamental ecosystem services provided by these communities. This study assessed the effects of organic matter removal and soil compaction on microbial community structures in different soil horizons 13 years after timber harvesting at the long‐term soil productivity site at Skulow Lake, British Columbia. A harvested stand was compared with an unmanaged forest stand. Ribosomal intergenic spacer profiles of bacteria, archaea and eukarya indicated significantly different community structures in the upper three soil horizons of the two stands, with differences decreasing with depth. Large‐scale sequencing of the ribosomal intergenic spacers coupled to small‐subunit ribosomal RNA genes allowed taxonomic identification of major microbial phylotypes affected by harvesting or varying among soil horizons. Actinobacteria and Gemmatimonadetes were the predominant phylotypes in the bacterial profiles, with the relative abundance of these groups highest in the unmanaged stand, particularly in the deeper soil horizons. Predominant eukaryal phylotypes were mainly assigned to known mycorrhizal and saprotrophic species of Basidiomycetes and Ascomycetes. Harvesting affected Basidiomycetes to a minor degree but had stronger effects on some Ascomycetes. Archaeal profiles had low diversity with only a few predominant crenarchaeal phylotypes whose abundance appeared to increase with depth. Detection of these effects 13 years after harvesting may indicate a long‐term change in processes mediated by the microbial community with important consequences for forest productivity. These effects warrant more comprehensive investigation of the effects of harvesting on the structure of forest soil microbial communities and the functional consequences.     

An alternative inverse PCR (IPCR) method to amplify DNA sequences flanking Tn5 transposon insertions

We have developed an alternative method to amplify DNA sequences flanking Tn5 transposon insertions. This method relies on the identical sequences of inverted terminal repeats, located at the 5' and 3' ends of Tn5, to determine the location and orientation of a transposon insertion within a restriction endonuclease fragment. From this information, PCR primers can be designed to selectively amplify by inverse PCR the DNA flanking one side of the transposon. This method avoids the problem of amplifying or cloning long sequences flanking Tn5. To demonstrate the applicability of this method, we generated Tn5 transposon mutants of Pseudomonas abietaniphila BKME-9 which no longer grew on dehydroabietic acid (DhA). The flanking sequence of one of the mutant (strain BKME-941) which accumulated 7-oxoDhA, was amplified.     

Structure-function analysis of herpes simplex virus type 1 gD and gH-gL: clues from gDgH chimeras

In alphaherpesviruses, glycoprotein B (gB), gD, gH, and gL are essential for virus entry. A replication-competent gL-null pseudorabies virus (PrV) (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014-3022, 1999) was shown to express a gDgH hybrid protein that could replace gD, gH, and gL in cell-cell fusion and null virus complementation assays. To study this phenomenon in herpes simplex virus type 1 (HSV-1), we constructed four gDgH chimeras, joining the first 308 gD amino acids to various gH N-terminal truncations. The chimeras were named for the first amino acid of gH at which each was truncated: 22, 259, 388, and 432. All chimeras were immunoprecipitated with both gD and gH antibodies to conformational epitopes. Normally, transport of gH to the cell surface requires gH-gL complex formation. Chimera 22 contains full-length gH fused to gD308. Unlike PrV gDgH, chimera 22 required gL for transport to the surface of transfected Vero cells. Interestingly, although chimera 259 failed to reach the cell surface, chimeras 388 and 432 exhibited gL-independent transport. To examine gD and gH domain function, each chimera was tested in cell-cell fusion and null virus complementation assays. Unlike PrV gDgH, none of the HSV-1 chimeras substituted for gL for fusion. Only chimera 22 was able to replace gH for fusion and could also replace either gH or gD in the complementation assay. Surprisingly, this chimera performed very poorly as a substitute for gD in the fusion assay despite its ability to complement gD-null virus and bind HSV entry receptors (HveA and nectin-1). Chimeras 388 and 432, which contain the same portion of gD as that in chimera 22, substituted for gD for fusion at 25 to 50% of wild-type levels. However, these chimeras functioned poorly in gD-null virus complementation assays. The results highlight the fact that these two functional assays are measuring two related but distinct processes.     

Antihypertensive effect of mechanism-based inhibition of renal arachidonic acid omega-hydroxylase activity

The cytochrome P-450 eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE) is a potent vasoconstrictor that is implicated in the regulation of blood pressure. The identification of selective inhibitors of renal 20-HETE formation for use in vivo would facilitate studies to determine the systemic effects of this eicosanoid. We characterized the acetylenic fatty acid sodium 10-undecynyl sulfate (10-SUYS) as a potent and selective mechanism-based inhibitor of renal 20-HETE formation. A single dose of 10-SUYS caused an acute reduction in mean arterial blood pressure in 8-wk-old spontaneously hypertensive rats. The decrease in mean arterial pressure was maximal 6 h after 10-SUYS treatment (17.9 +/- 3.2 mmHg; P < 0.05), and blood pressure returned to baseline levels within 24 h after treatment. Treatment with 10-SUYS was associated with a decrease in urinary 20-HETE formation in vivo and attenuation of the vasoconstrictor response of renal interlobar arteries to ANG II in vitro. These results provide further evidence that 20-HETE plays an important role in the regulation of blood pressure in the spontaneously hypertensive rat.     

alpha(1)-Adrenoceptor agonists: the identification of novel alpha(1A )subtype selective 2'-heteroaryl-2-(phenoxymethyl)imidazolines

Novel 2'-heteroaryl-2-(phenoxymethyl)imidazolines have been identified as potent agonists of the cloned human alpha(1)-adrenoceptors in vitro. The nature of the 2'-heteroaryl group can have significant effects on the potency, efficacy, and subtype selectivity in this series. alpha(1A) Subtype selective agonists have been identified.