The Arabidopsis cullin AtCUL1 is modified by the ubiquitin-related protein RUB1

The ubiquitin-like protein RUB1 is conjugated to target proteins by a mechanism similar to that of ubiquitin conjugation. Genetic studies in Arabidopsis thaliana have implicated the RUB-conjugation pathway in auxin response. The first step in the pathway is RUB activation by a bipartite enzyme composed of the AXR1 and ECR1 proteins. Ubiquitin activation is an ATP-dependent process that involves the formation of an AMP-ubiquitin intermediate. Here we show that RUB activation by AXR1-ECR1 also involves formation of an AMP-RUB intermediate and that this reaction is catalyzed by the ECR1 subunit alone. In addition, we identified an Arabidopsis protein called RCE1 that is a likely RUB-conjugating enzyme. RCE1 works together with AXR1-ECR1 to promote formation of a stable RUB conjugate with the Arabidopsis cullin AtCUL1 in vitro. Using a tagged version of RUB1, we show that this modification occurs in vivo. Because AtCUL1 is a component of the ubiquitin protein ligase SCFTIR1, a complex that also functions in auxin response, we propose that RUB modification of AtCUL1 is important for auxin response.

Inhibition of the ubiquitin-proteasome system in Alzheimer's disease

Alzheimer's disease is the most common cause of dementia in the elderly. Although several genetic defects have been identified in patients with a family history of this disease, the majority of cases involve individuals with no known genetic predisposition. A mutant form of ubiquitin, termed Ub+1, has been selectively observed in the brains of Alzheimer's patients, including those with nonfamilial Alzheimer's disease, but it has been unclear why Ub+1 expression should be deleterious. Here we show that Ub+1 is an efficient substrate for polyubiquitination in vitro and in transfected human cells. The resulting polyubiquitin chains are refractory to disassembly by deubiquitinating enzymes and potently inhibit the degradation of a polyubiquitinated substrate by purified 26S proteasomes. Thus, expression of Ub+1 in aging brain could result in dominant inhibition of the Ub-proteasome system, leading to neuropathologic consequences.

The ubiquitin-proteasome system in retinal health and disease

The ubiquitin–proteasome system (UPS) is the main intracellular pathway for modulated protein turnover, playing an important role in the maintenance of cellular homeostasis. It also exerts a protein quality control through degradation of oxidized, mutant, denatured, or misfolded proteins and is involved in many biological processes where protein level regulation is necessary. This system allows the cell to modulate its protein expression pattern in response to changing physiological conditions and provides a critical protective role in health and disease. Impairments of UPS function in the central nervous system (CNS) underlie an increasing number of genetic and idiopathic diseases, many of which affect the retina. Current knowledge on the UPS composition and function in this tissue, however, is scarce and dispersed. This review focuses on UPS elements reported in the retina, including ubiquitinating and deubiquitinating enzymes (DUBs), and alternative proteasome assemblies. Known and inferred roles of protein ubiquitination, and of the related, SUMO conjugation (SUMOylation) process, in normal retinal development and adult homeostasis are addressed, including modulation of the visual cycle and response to retinal stress and injury. Additionally, the relationship between UPS dysfunction and human neurodegenerative disorders affecting the retina, including Alzheimer's, Parkinson's, and Huntington's diseases, are dealt with, together with numerous instances of retina-specific illnesses with UPS involvement, such as retinitis pigmentosa, macular degenerations, glaucoma, diabetic retinopathy (DR), and aging-related impairments. This information, though still basic and limited, constitutes a suitable framework to be expanded in incoming years and should prove orientative toward future therapy design targeting sight-affecting diseases with a UPS underlying basis.

Small Ubiquitin-related Modifier (SUMO)-1 promotes glycolysis in hypoxia

Under conditions of hypoxia, most eukaryotic cells undergo a shift in metabolic strategy, which involves increased flux through the glycolytic pathway. Although this is critical for bioenergetic homeostasis, the underlying mechanisms have remained incompletely understood. Here, we report that the induction of hypoxia-induced glycolysis is retained in cells when gene transcription or protein synthesis are inhibited suggesting the involvement of additional post-translational mechanisms. Post-translational protein modification by the small ubiquitin related modifier-1 (SUMO-1) is induced in hypoxia and mass spectrometric analysis using yeast cells expressing tap-tagged Smt3 (the yeast homolog of mammalian SUMO) revealed hypoxia-dependent modification of a number of key glycolytic enzymes. Overexpression of SUMO-1 in mammalian cancer cells resulted in increased hypoxia-induced glycolysis and resistance to hypoxia-dependent ATP depletion. Supporting this, non-transformed cells also demonstrated increased glucose uptake upon SUMO-1 overexpression. Conversely, cells overexpressing the de-SUMOylating enzyme SENP-2 failed to demonstrate hypoxia-induced glycolysis. SUMO-1 overexpressing cells demonstrated focal clustering of glycolytic enzymes in response to hypoxia leading us to hypothesize a role for SUMOylation in promoting spatial re-organization of the glycolytic pathway. In summary, we hypothesize that SUMO modification of key metabolic enzymes plays an important role in shifting cellular metabolic strategies toward increased flux through the glycolytic pathway during periods of hypoxic stress. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Sulfur transfer and activation by ubiquitin-like modifier system Uba4•Urm1 link protein urmylation and tRNA thiolation in yeast

Urm1 is a unique dual-function member of the ubiquitin protein family and conserved from yeast to man. It acts both as a protein modifier in ubiquitin-like urmylation and as a sulfur donor for tRNA thiolation, which in concert with the Elongator pathway forms 5-methoxy-carbonyl-methyl-2-thio (mcm5s2) modified wobble uridines (U34) in anticodons. Using Saccharomyces cerevisiae as a model to study a relationship between these two functions, we examined whether cultivation temperature and sulfur supply previously implicated in the tRNA thiolation branch of the URM1 pathway also contribute to proper urmylation. Monitoring Urm1 conjugation, we found urmylation of the peroxiredoxin Ahp1 is suppressed either at elevated cultivation temperatures or under sulfur starvation. In line with this, mutants with sulfur transfer defects that are linked to enzymes (Tum1, Uba4) required for Urm1 activation by thiocarboxylation (Urm1-COSH) were found to maintain drastically reduced levels of Ahp1 urmylation and mcm5s2U34 modification. Moreover, as revealed by site specific mutagenesis, the Stransfer rhodanese domain (RHD) in the E1-like activator (Uba4) crucial for Urm1-COSH formation is critical but not essential for protein urmylation and tRNA thiolation. In sum, sulfur supply, transfer and activation chemically link protein urmylation and tRNA thiolation. These are features that distinguish the ubiquitin-like modifier system Uba4•Urm1 from canonical ubiquitin family members and will help elucidate whether, in addition to their mechanistic links, the protein and tRNA modification branches of the URM1 pathway may also relate in function to one another.

Digestive Peptidase Evolution In Holometabolous Insects Led To A Divergent Group Of Enzymes In Lepidoptera.

Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic l-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

A Leucine-rich Diet Modulates The Tumor-induced Down-regulation Of The Mapk/erk And Pi3k/akt/mtor Signaling Pathways And Maintains The Expression Of The Ubiquitin-proteasome Pathway In The Placental Tissue Of Nmri Mice.

Placental tissue injury is concomitant with tumor development. We investigated tumor-driven placental damage by tracing certain steps of the protein synthesis and degradation pathways under leucine-rich diet supplementation in MAC16 tumor-bearing mice. Cell signaling and ubiquitin-proteasome pathways were assessed in the placental tissues of pregnant mice, which were distributed into three groups on a control diet (pregnant control, tumor-bearing pregnant, and pregnant injected with MAC-ascitic fluid) and three other groups on a leucine-rich diet (pregnant, tumor-bearing pregnant, and pregnant injected with MAC-ascitic fluid). MAC tumor growth down-regulated the cell-signaling pathways of the placental tissue and decreased the levels of IRS-1, Akt/PKB, Erk/MAPK, mTOR, p70S6K, STAT3, and STAT6 phosphorylated proteins, as assessed by the multiplex Millipore Luminex assay. Leucine supplementation maintained the levels of these proteins within the established cell-signaling pathways. In the tumor-bearing group (MAC) only, the placental tissue showed increased PC5 mRNA expression, as assessed by quantitative RT-PCR, decreased 19S and 20S protein expression, as assessed by Western blot analysis, and decreased placental tyrosine levels, likely reflecting up-regulation of the ubiquitin-proteasome pathway. Similar effects were found in the pregnant injected with MAC-ascitic fluid group, confirming that the effects of the tumor were mimicked by MAC-ascitic fluid injection. Although tumor progression occurred, the degradation pathway-related protein levels were modulated under leucine-supplementation conditions. In conclusion, tumor evolution reduced the protein expression of the cell-signaling pathway associated with elevated protein degradation, thereby jeopardizing placental activity. Under the leucine-rich diet, the impact of cancer on placental function could be minimized by improving the cell-signaling activity and reducing the proteolytic process.

Production of Cellulolytic Enzymes by Anaerobic Fungi Cultivated in Different Conditions

The present paper studies the influence of different nutrients for the production of two cellulolytic enzymes: endo beta-1.4 glucanase and exo beta-1.4 glucanase by anaerobic fungi taken from cow rumen, that were fed a diet of corn silage and Brachiaria decumbens grass hay. During the enzymatic degradation assays, it was observed that the addition of some essential nutrients in the formulation of the culture medium contributed positively in the cellulolytic enzyme production, with exception of riboflavin. Such results contributed in the establishment of an effective method for the evaluation of enzymatic activities in anaerobic fibrolytic fungi. In this work, nutrients added to enrich the culture medium have successfully proven that they can be used as inoculating agents (inductors) in diets rich in ensilage with law nutritive value.

Use of Cassava Peel as Carbon Source for Production of Amylolytic Enzymes by Aspergillus niveus

Aspergillus niveus produced high levels of alpha-amylase and glucoamylase in submerged fermentation using the agricultural residue cassava peel as a carbon source. In static conditions, the amylase production was substantially greater than in the agitated condition. The optimized culture conditions were initially at pH 5.0, 35 degrees C during 48 hours. Amylolytic activity was still improved (50%) with a mixture of cassava peel and soluble starch in the proportion 1:1 (w/w). The crude extract exhibited temperature and pH optima approximately 70 degrees C and 4.5, respectively. Amylase activity was stable for 1 h at 60 degrees C, and at pH values between 3.0 and 7.0. The enzyme hydrolysed preferentially maltose, starch, penetrose, amylose, isomaltose, maltotriose, glycogen and amylopectin, and not hydrolysed cyclodextrin (alpha and beta), trehalose and sucrose. In the first hour of reaction on soluble starch, the hydrolysis products were glucose and maltose, but after two hours of hydrolysis, glucose was the unique product formed, confirming the presence in the crude extract of an alpha-amylase and a glucoamylase.

Sequential production of amylolytic and lipolytic enzymes by bacterium strain isolated from petroleum contaminated soil

Amylases and lipases are highly demanded industrial enzymes in various sectors such as food, pharmaceuticals, textiles, and detergents. Amylases are of ubiquitous occurrence and hold the maximum market share of enzyme sales. Lipases are the most versatile biocatalyst and bring about a range of bioconversion reactions such as hydrolysis, inter-esterification, esterification, alcoholysis, acidolysis, and aminolysis. The objective of this work was to study the feasibility for amylolitic and lipolytic production using a bacterium strain isolated from petroleum contaminated soil in the same submerged fermentation. This was a sequential process based on starch and vegetable oils feedstocks. Run were performed in batchwise using 2% starch supplemented with suitable nutrients and different vegetable oils as a lipase inducers. Fermentation conditions were pH 5.0; 30 degrees C, and stirred speed (200 rpm). Maxima activities for amyloglucosidase and lipase were, respectively, 0.18 and 1,150 U/ml. These results showed a promising methodology to obtain both enzymes using industrial waste resources containing vegetable oils.

Evaluation of hexose and pentose in pre-cultivation of Candida guilliermondii on the key enzymes for xylitol production in sugarcane hemicellulosic hydrolysate

The evaluation of hexose and pentose in pre-cultivation of Candida guilliermondii FTI 20037 yeast on xylose reductase (XR) and xylitol dehydrogenase (XDH) enzymes activities was performed during fermentation in sugarcane bagasse hemicellulosic hydrolysate. The xylitol production was evaluated by using cells previously growth in 30.0 gl(-1) xylose, 30.0 gl(-1) glucose and in both sugars mixture (30.0 gl(-1) xylose and 2.0 gl(-1) glucose). The vacuum evaporated hydrolysate (80 gl(-1)) was detoxificated by ion exchange resin (A-860S; A500PS and C-150-Purolite(A (R))). The total phenolic compounds and acetic acid were 93.0 and 64.9%, respectively, removed by the resin hydrolysate treatment. All experiments were carried out in Erlenmeyer flasks at 200 rpm, 30A degrees C. The maximum XR (0.618 Umg (Prot) (-1) ) and XDH (0.783 Umg (Prot) (-1) ) enzymes activities was obtained using inoculum previously growth in both sugars mixture. The highest cell concentration (10.6 gl(-1)) was obtained with inoculum pre-cultivated in the glucose. However, the xylitol yield and xylitol volumetric productivity were favored using the xylose as carbon source. In this case, it was observed maximum xylose (81%) and acetic acid (100%) consumption. It is very important to point out that maximum enzymatic activities were obtained when the mixture of sugars was used as carbon source of inoculum, while the highest fermentative parameters were obtained when xylose was used.

Use of activated charcoal and ion-exchange resin to cleaN up and concentrate enzymes in extracts from biodegraded wood.

Use of activated charcoal and ion-exchange resin to cleaN up and concentrate enzymes in extracts from biodegraded wood. Ceriporiopsis subvermispora was used for the biodegradation of Eucalyptus grandis chips in the presence or absence of co-substrates (glucose and corn steep liquor) during 7, 14 and 28 days. Afterwards, the biodegraded chips were extracted with 50 mM sodium acetate buffer (pH 5.5) supplemented with 0.01% Tween 60. High activities of manganese peroxidases (MnPs) were observed in all the extracts, both in the absence (430, 765 and 896 UI kg(-1) respectively) and in the presence of co-substrates (1,013; 2,066 and 2,323 UI kg(-1) respectively). The extracts presented a high ratio between absorbances at 280 and 405 nm, indicating a strong abundance of aromatic compounds derived from lignin over heme-peroxidases. Adsorption into activated charcoal showed to be an adequate strategy to reduce the absorbance at 280 urn in all the extracts. Moreover, it allowed to maximize the capacity of an anion exchange resin bed (DEAE-Sepharose) used to concentrate the MnPs present in the extracts. It was concluded that the use of activated charcoal followed by adsorption into DEAE Sepharose is a strategy that can be used to concentrate MnPs in extracts obtained during the biodegradation of E. grandis by C. subvermispora.

Linoleic acid peroxidation and lignin degradation by enzymes produced by Ceriporiopsis subvermispora grown on wood or in submerged liquid cultures

Ceriporiopsis subvermispora is a white-rot fungus used in biopulping processes and seems to use the fatty acid peroxidation reactions initiated by manganese-peroxidase (MnP) to start lignin degradation. The present work shows that C. subvermispora was able to peroxidize unsaturated fatty acids during wood biotreatment under biopulping conditions. In vitro assays showed that the extent of linoleic acid peroxidation was positively correlated with the level of MnP recovered from the biotreated wood chips. Milled wood was treated in vitro by partially purified MnP and linoleic acid. UV spectroscopy and size exclusion chromatography (SEC) showed that soluble compounds similar to lignin were released from the milled wood. SEC data showed a broad elution profile compatible with low molar mass lignin fractions. MnP-treated milled wood was analyzed by thioacidolysis. The yield of thioacidolysis monomers recovered from guaiacyl and syringyl units decreased by 33% and 20% in MnP-treated milled wood, respectively. This has suggested that lignin depolymerization reactions have occurred during the MnP/linoleic acid treatment. (C) 2009 Elsevier Inc. All rights reserved.

Bioinsecticidal activity of Talisia esculenta reserve protein on growth and serine digestive enzymes during larval development of Anticarsia gemmatalis

Plants synthesize a variety of molecules to defend themselves against an attack by insects. Talisin is a reserve protein from Talisia esculenta seeds, the first to be characterized from the family Sapindaceae. In this study, the insecticidal activity of Talisin was tested by incorporating the reserve protein into an artificial diet fed to the velvetbean caterpillar Anticarsia gemmatalis, the major pest of soybean crops in Brazil. At 1.5% (w/w) of the dietary protein, Talisin affected larval growth, pupal weight, development and mortality, adult fertility and longevity, and produced malformations in pupae and adult insects. Talisin inhibited the trypsin-like activity of larval midgut homogenates. The trypsin activity in Talisin-fed larvae was sensitive to Talisin, indicating that no novel protease-resistant to Talisin was induced in Talisin-fed larvae. Affinity chromatography showed that Talisin bound to midgut proteinases of the insect A. gemmatalis, but was resistant to enzymatic digestion by these larval proteinases. The transformation of genes coding for this reserve protein could be useful for developing insect resistant crops. (C) 2010 Elsevier Inc. All rights reserved.

The isolation of antioxidant enzymes from mature tomato (cv. Micro-Tom) plants

The activity of catalase (CAT), guaiacol peroxidase (GPOX), ascorbate peroxidase (APX), glutathione reductase (GR), and the isoenzymes of superoxide dismutase (SOD) were determined in the organs of tomato (Lycopersicon esculentum) cultivar Micro-Tom after 104 days of development. The total activities of CAT, GPOX, and GR were higher in the stem than in others tissues, whereas the stem exhibited the lowest APX activity. Activity staining analysis following gel electrophoresis revealed the existence of four SOD isoenzymes in leaves, three in fruits, but only two in the roots and stems. This characterization is essential for an investigation into the effect of abiotic and biotic stresses on the oxidative stress responses by this plant model system.