Long-term elevation of β-hydroxybutyrate in dairy cows through infusion: effects on feed intake, milk production, and metabolism.

Elevation of ketone bodies in dairy cows frequently occurs in early lactation, usually concomitantly with a lack of energy and glucose. The objective of this study was to induce an elevated plasma β-hydroxybutyrate (BHBA) concentration over 48 h in mid-lactating dairy cows (i.e., during a period of positive energy balance and normal glucose plasma concentrations). Effects of BHBA infusion on feed intake, metabolism, and performance were investigated. Thirteen cows were randomly assigned to 1 of 2 infusion groups, including an intravenous infusion with Na-dl-β-OH-butyrate (1.7 mol/L) to achieve a plasma concentration of 1.5 to 2.0 mmol/L of BHBA (HyperB; n=5), or an infusion of 0.9% saline solution (control; n=8). Blood was sampled before and hourly during the 48 h of infusion. In the liver, mRNA transcripts related to gluconeogenesis (pyruvate carboxylase, glucose 6-phosphatase, mitochondrial phosphoenolpyruvate carboxykinase), phosphofructokinase, pyruvate dehydrogenase complex, and fatty acid synthesis (acetyl-coenzyme A carboxylase, fatty acid synthase) were measured by real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase and ubiquitin were used as housekeeping genes. Changes (difference between before and after 48-h infusion) during the infusion period were evaluated by ANOVA with treatment as fixed effect, and area under the curve of variables was calculated on the second day of experiment. The plasma BHBA concentration in HyperB cows was 1.74 ± 0.02 mmol/L (mean ± SE) compared with 0.59 ± 0.02 mmol/L for control cows. The change in feed intake, milk yield, and energy corrected milk did not differ between the 2 experimental groups. Infusion of BHBA reduced the plasma glucose concentration (3.47 ± 0.11 mmol/L) in HyperB compared with control cows (4.11 ± 0.08 mmol/L). Plasma glucagon concentration in HyperB was lower than the control group. All other variables measured in plasma were not affected by treatment. In the liver, changes in mRNA abundance for the selected genes were similar between 2 groups. Results demonstrate that intravenous infusion of BHBA decreased plasma glucose concentration in dairy cows, but this decrease could not be explained by alterations in insulin concentrations or key enzymes related to gluconeogenesis. Declined glucose concentration is likely functionally related to decreased plasma glucagon concentration.

Induction of cytochrome P450 enzymes in primary equine hepatocyte culture

In this study, we established cell culture conditions for primary equine hepatocytes allowing cytochrome P450 enzyme (CYP) induction experiments. Hepatocytes were isolated after a modified method of Bakala et al. (2003) and cultivated on collagen I coated plates. Three different media were compared for their influence on morphology, viability and CYP activity of the hepatocytes. CYP activity was evaluated with the fluorescent substrate 7-benzyloxy-4-trifluoromethylcoumarin. Induction experiments were carried out with rifampicin, dexamethasone or phenobarbital. Concentration-response curves for induction with rifampicin were created. Williams' medium E showed the best results on morphology and viability of the hepatocytes and was therefore used for the following induction experiments. Cells cultured in Dulbecco's Modified Eagle Medium were not inducible. Incubation with rifampicin increased the CYP activity in two different hepatocyte preparations in a dose dependent manner (EC50=1.20 μM and 6.06 μM; Emax=4.1- and 3.4-fold induction). No increase in CYP activity was detected after incubation with dexamethasone or phenobarbital. The hepatocyte culture conditions established in this study proved to be valuable for investigation of the induction of equine CYPs. In further studies, other equine drugs can be evaluated for CYP induction with this in vitro system.

The ubiquitin-proteasome system is necessary for long-term synaptic depression in Aplysia.

The neuropeptide Phe-Met-Arg-Phe-NH(2) (FMRFa) can induce transcription-dependent long-term synaptic depression (LTD) in Aplysia sensorimotor synapses. We investigated the role of the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to presynaptic inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by CREB2, which is generally regarded as a transcription repressor. Binding of CREB2 to the promoter region of ap-uch was accompanied by histone hyperacetylation, suggesting that CREB2 cannot only inhibit but also promote gene expression. CREB2 was phosphorylated after FMRFa, and blocking phospho-CREB2 blocked LTD. In addition to changes in the expression of ap-uch, the synaptic vesicle-associated protein synapsin was downregulated in LTD in a proteasome-dependent manner. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions.

THE UBIQUITIN LIGASE UBE4B IS REQUIRED FOR EFFICIENT EPIDERMAL GROWTH FACTOR RECEPTOR DEGRADATION

The length of time that integral membrane proteins reside on the plasma membrane is regulated by endocytosis, a process that can inactivate these proteins by removing them from the membrane and may ultimately result in their degradation. Proteins are internalized and pass through multiple distinct intracellular compartments where targeting decisions determine their fate. Membrane proteins initially enter early endosomes, and subsequently late endosomes/multivesicular bodies (MVBs), before being degraded in the lysosome. The MVB is a subset of late endosomes characterized by the appearance of small vesicles in its luminal compartment. These vesicles contain cargo proteins sorted from the limiting membrane of the MVB. Proteins not sorted into luminal vesicles remain on the MVB membrane, from where they may be recycled back to the plasma membrane. In the case of receptor tyrosine kinases (RTKs), such as epidermal growth factor (EGF) receptor, this important sorting step determines whether a protein returns to the surface to participate in signaling, or whether its signaling properties are inactivated through its degradation in the lysosome. Hrs is a protein that resides on endosomes and is known to recruit sorting complexes that are vital to this sorting step. These sorting complexes are believed to recognize ubiquitin as sorting signals. However, the link between MVB sorting machinery and the ubiquitination machinery is not known. Recently, Hrs was shown to recruit and bind an E3 ubiquitin ligase, UBE4B, to endosomes. In an assay that is able to measure cargo movement, the disruption of the Hrs-UBE4B interaction showed impaired sorting of EGF receptor into MVBs. My hypothesis is that UBE4B may be the connection between MVB sorting and ubiquitination. This study addresses the role of UBE4B in the trafficking and ubiquitination of EGF receptor. I created stable cell lines that either overexpresses UBE4B or expresses a UBE4B with no ligase activity. Levels of EGF receptor were analyzed after certain periods of ligand-induced receptor internalization. I observed that higher expression levels of UBE4B correspond to increased degradation of EGF receptor. In an in vitro ubiquitination assay, I also determined that UBE4B mediates the ubiquitination of EGF receptor. These data suggest that UBE4B is required for EGFR degradation specifically because it ubiquitinates the receptor allowing it to be sorted into the internal vesicles of MVBs and subsequently degraded in lysosomes.

ARTEMIS INTERACTS WITH THE CUL4A UBIQUITIN E3 LIGASE COMPLEX AND REGULATES THE CELL CYCLE PROGRESSION

Artemis, a member of the SNM1 gene family, is one of the six known components of the non-homologous end joining pathway. It is a multifunctional phospho-protein that has been shown to be modified by the phosphatidylinositol 3-kinases (PIKs) DNA-PKcs, ATM and ATR in response to a variety of cellular stresses. Artemis has important roles in V(D)J recombination, DNA double strand breaks repair and damage-induced cell-cycle checkpoint regulation. The detailed mechanism by which Artemis mediates its functions in these cellular pathways needs to be further elucidated. My work presented here demonstrates a new function for Artemis in cell cycle regulation as a component of Cullin-based E3 ligase complex. I show that Artemis interacts with Cul4A-DDB1 ligase complex via a direct interaction with the substrate-specific receptor DDB2, and deletion mapping analysis shows that part of the Snm1 domain of Artemis is responsible for this interaction. Additionally, Artemis also interacts with p27, a substrate of Cul4A-DDB1 complex, and both DDB2 and Artemis are required for the degradation of p27 mediated by this complex. Furthermore, I show that the regulation of p27 by Artemis and DDB2 is critical for cell cycle progression in normally proliferating cells and in response to serum withdrawal. Finally, I provide evidence showing that Artemis may be also a part of other Cullin-based E3 ligase complexes, and it has a role in controlling p27 levels in response to different cellular stress, such as UV irradiation. These findings suggest a novel pathway to regulate p27 protein level and define a new function for Artemis as an effector of Cullin-based E3-ligase mediated ubiquitylation, and thus, a cell cycle regulator in proliferating cells.

COP 9 SIGNALOSOME SUBUNIT 6 STABILIZES COP1, A NOVEL E3 UBIQUITIN LIGASE FOR 14-3-3σ

14-3-3σ, a gene upregulated by p53 in response to DNA damage, exists as part of a positive-feedback loop which activates p53 and is a human cancer epithelial marker downregulated in various cancer types. 14-3-3σ levels are critical for maintaining p53 activity in response to DNA damage and regulating signal mediator such as Akt. Here, we identify Mammalian Constitutive Photomorphogenic 1 (COP1) as a novel E3 ubiquitin ligase for targeting 14-3-3σ through proteasome degradation. We show for the first time that COP9 signalosome subunit 6 (CSN6) associates with COP1 and is involved in 14-3-3σ ubiquitin-mediated degradation. Mechanistic studies show that CSN6 expression leads to stabilization of COP1 through reducing COP1 self-ubiquitination and decelerating COP1’s turnover rate. We also show that CSN6-mediated 14-3-3σ ubiquitination is compromised when COP1 is knocked down. Thus, CSN6 mediates 14-3-3σ ubiquitination through enhancing COP1 stability. Subsequently, we show that CSN6 causes 14-3-3σ downregulation, thereby activating Akt and promoting cell survival by suppressing FOXO, an Akt target, transcriptional activity. Also, CSN6 overexpression leads to increased cell growth, transformation and promotes tumorigenicity. Significantly, 14-3-3σ expression can correct the abnormalities mediated by CSN6 expression. These data suggest that the CSN6-COP1 axis is involved in 14-3-3σ degradation, and that deregulation of this axis will promote cell growth and tumorigenicity.

Genetic variation in genes for the xenobiotic-metabolizing enzymes CYP1A1, EPHX1, GSTM1, GSTT1, and GSTP1 and susceptibility to colorectal cancer in Lynch syndrome.

Individuals with Lynch syndrome are predisposed to cancer due to an inherited DNA mismatch repair gene mutation. However, there is significant variability observed in disease expression likely due to the influence of other environmental, lifestyle, or genetic factors. Polymorphisms in genes encoding xenobiotic-metabolizing enzymes may modify cancer risk by influencing the metabolism and clearance of potential carcinogens from the body. In this retrospective analysis, we examined key candidate gene polymorphisms in CYP1A1, EPHX1, GSTT1, GSTM1, and GSTP1 as modifiers of age at onset of colorectal cancer among 257 individuals with Lynch syndrome. We found that subjects heterozygous for CYP1A1 I462V (c.1384A>G) developed colorectal cancer 4 years earlier than those with the homozygous wild-type genotype (median ages, 39 and 43 years, respectively; log-rank test P = 0.018). Furthermore, being heterozygous for the CYP1A1 polymorphisms, I462V and Msp1 (g.6235T>C), was associated with an increased risk for developing colorectal cancer [adjusted hazard ratio for AG relative to AA, 1.78; 95% confidence interval, 1.16-2.74; P = 0.008; hazard ratio for TC relative to TT, 1.53; 95% confidence interval, 1.06-2.22; P = 0.02]. Because homozygous variants for both CYP1A1 polymorphisms were rare, risk estimates were imprecise. None of the other gene polymorphisms examined were associated with an earlier onset age for colorectal cancer. Our results suggest that the I462V and Msp1 polymorphisms in CYP1A1 may be an additional susceptibility factor for disease expression in Lynch syndrome because they modify the age of colorectal cancer onset by up to 4 years.

Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure.

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.

The ubiquitin-proteasome system is necessary for long-term synaptic depression in Aplysia.

The neuropeptide Phe-Met-Arg-Phe-NH(2) (FMRFa) can induce transcription-dependent long-term synaptic depression (LTD) in Aplysia sensorimotor synapses. We investigated the role of the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to presynaptic inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by CREB2, which is generally regarded as a transcription repressor. Binding of CREB2 to the promoter region of ap-uch was accompanied by histone hyperacetylation, suggesting that CREB2 cannot only inhibit but also promote gene expression. CREB2 was phosphorylated after FMRFa, and blocking phospho-CREB2 blocked LTD. In addition to changes in the expression of ap-uch, the synaptic vesicle-associated protein synapsin was downregulated in LTD in a proteasome-dependent manner. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions.

Identification and characterization of genes encoding phospholipid biosynthetic enzymes of {\it Saccharomyces cerevisiae\/}

Phospholipids are the major component of cellular membranes. In addition to its structural role, phospholipids play an active and diverse role in cellular processes. The goal of this study is to identify the genes involved in phospholipid biosynthesis in a model eukaryotic system, Saccharomyces cerevisiae. We have focused on the biosynthetic steps localized in the inner mitochondrial membrane; hence, the identification of the genes encoding phosphatidylserine decarboxylase (PSD1), cardiolipin synthase (CLS1), and phosphatidylglycerophosphate synthase (PGS1).^ The PSD1 gene encoding a phosphatidylserine decarboxylase was cloned by complementation of a conditional lethal mutation in the homologous gene in Escherichia coli strain EH150. Overexpression of the PSD1 gene in wild type yeast resulted in 20-fold amplification of phosphatidylserine decarboxylase activity. Disruption of the PSD1 gene resulted in 20-fold reduction of decarboxylase activity, but the PSD1 null mutant exhibited essentially normal phenotype. These results suggest that yeast has a second phosphatidylserine decarboxylation activity.^ Cardiolipin is the major anionic phospholipid of the inner mitochondrial membrane. It is thought to be an essential component of many biochemical functions. In eukaryotic cells, cardiolipin synthase catalyzes the final step in the synthesis of cardiolipin from phosphatidylglycerol and CDP-diacylglycerol. We have cloned the gene CLS1. Overexpression of the CLS1 gene product resulted in significantly elevated cardiolipin synthase activity, and disruption of the CLS1 gene, confirmed by PCR and Southern blot analysis, resulted in a null mutant that was viable and showed no petite phenotype. However, phospholipid analysis showed undetectable cardiolipin level and an accumulation of phosphatidylglycerol. These results support the conclusion that CLS1 encodes the cardiolipin synthase of yeast and that normal levels of cardiolipin are not absolutely essential for survival of the cell.^ Phosphatidylglycerophosphate (PGP) synthase catalyzes the synthesis of PGP from CDP-diacylglycerol and glycerol-3-phosphate and functions as the committal and rate limiting step in the biosynthesis of cardiolipin. We have identified the PGS1 gene as encoding the PGP synthase. Overexpression of the PGS1 gene product resulted in over 15-fold increase in in vitro PGP synthase activity. Disruption of the PGS1 gene in a haploid strain of yeast, confirmed by Southern blot analysis, resulted in a null mutant strain that was viable but had significantly altered phenotypes, i.e. inability to grow on glycerol and at $37\sp\circ$C. These cells showed over a 10-fold decrease in PGP synthase activity and a decrease in both phosphatidylglycerol and cardiolipin levels. These results support the conclusion that PGS1 encodes the PGP synthase of yeast and that neither phosphatidylglycerol nor cardiolipin are absolutely essential for survival of the cell. ^

Genetic and microbial factors modulating the ubiquitin proteasome system in inflammatory bowel disease

OBJECTIVE: Altered microbiota composition, changes in immune responses and impaired intestinal barrier functions are observed in IBD. Most of these features are controlled by proteases and their inhibitors to maintain gut homeostasis. Unrestrained or excessive proteolysis can lead to pathological gastrointestinal conditions. The aim was to validate the identified protease IBD candidates from a previously performed systematic review through a genetic association study and functional follow-up. DESIGN: We performed a genetic association study in a large multicentre cohort of patients with Crohn's disease (CD) and UC from five European IBD referral centres in a total of 2320 CD patients, 2112 UC patients and 1796 healthy controls. Subsequently, we did an extensive functional assessment of the candidate genes to explore their causality in IBD pathogenesis. RESULTS: Ten single nucleotide polymorphisms (SNPs) in four genes were significantly associated with CD: CYLD, USP40, APEH and USP3. CYLD was the most significant gene with the intronically located rs12324931 the strongest associated SNP (pFDR=1.74e-17, OR=2.24 (1.83 to 2.74)). Five SNPs in four genes were significantly associated with UC: USP40, APEH, DAG1 and USP3. CYLD, as well as some of the other associated genes, is part of the ubiquitin proteasome system (UPS). We therefore determined if the IBD-associated adherent-invasive Escherichia coli (AIEC) can modulate the UPS functioning. Infection of intestinal epithelial cells with the AIEC LF82 reference strain modulated the UPS turnover by reducing poly-ubiquitin conjugate accumulation, increasing 26S proteasome activities and decreasing protein levels of the NF-κB regulator CYLD. This resulted in IκB-α degradation and NF-κB activation. This activity was very important for the pathogenicity of AIEC since decreased CYLD resulted in increased ability of AIEC LF82 to replicate intracellularly. CONCLUSIONS: Our results reveal the UPS, and CYLD specifically, as an important contributor to IBD pathogenesis, which is favoured by both genetic and microbial factors.

Ubiquitin-specific protease USP2-45 acts as a molecular switch to promote α2δ-1-induced downregulation of Ca v1.2 channels

Availability of voltage-gated calcium channels (Cav) at the plasma membrane is paramount to maintaining the calcium homeostasis of the cell. It is proposed that the ubiquitylation/de-ubiquitylation balance regulates the density of ion channels at the cell surface. Voltage-gated calcium channels Cav1.2 have been found to be ubiquitylated under basal conditions both in vitro and in vivo. In a previous study, we have shown that Cav1.2 channels are ubiquitylated by neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4-1) ubiquitin ligases, but the identity of the counterpart de-ubiquitylating enzyme remained to be elucidated. Regarding sodium and potassium channels, it has been reported that the action of the related isoform Nedd4-2 is counteracted by the ubiquitin-specific protease (USP) 2-45. In this study, we show that USP 2-45 also de-ubiquitylates Cav channels. We co-expressed USPs and Cav1.2 channels together with the accessory subunits β2 and α2δ-1, in tsA-201 and HEK-293 mammalian cell lines. Using whole-cell current recordings and surface biotinylation assays, we show that USP2-45 specifically decreases both the amplitude of Cav currents and the amount of Cav1.2 subunits inserted at the plasma membrane. Importantly, co-expression of the α2δ-1 accessory subunit is necessary to support the effect of USP2-45. We further show that USP2-45 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits. Remarkably, α2δ-1, but not Cav1.2 nor β2, co-precipitated with USP2-45. These results suggest that USP2-45 binding to α2δ-1 promotes the de-ubiquitylation of both Cav1.2 and α2δ-1 subunits, in order to regulate the expression of Cav1.2 channels at the plasma membrane.

Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.

The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A94 since the CYP activity was significantly enhanced with co-expressed POR.

Two enzymes responsible for the formation of herbivore-induced volatiles of maize, the methyltransferase AAMT1 and the terpene synthase TPS23, are regulated by a similar signal transduction pathway

Herbivore-induced volatiles play an important role in the indirect defense of plants. After herbivore damage, volatiles are released from the plant and can attract herbivore enemies that protect the plant from additional damage. The herbivore-induced volatile blend is complex and usually consists of mono- and sesquiterpenes, aromatic compounds, and indole. Although these classes of compounds are generally produced at different times after herbivore damage, the release of the terpene (E)-β-caryophyllene and the aromatic ester methyl anthranilate appear to be tightly coordinated. We have studied the herbivore induction patterns of two terpene synthases from Zea mays L. (Poaceae), TPS23 and TPS10, as well as S-adenosyl-L-methionine:anthranilic acid carboxyl methyltransferases (AAMT1), which are critical for the production of terpenes and anthranilate compounds, respectively. The transcript levels of tps23 and aamt1 displayed the same kinetics after damage by the larvae of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae), and showed the same organ-specific and haplotype-specific expression patterns. Despite its close functional relation to TPS23, the terpene synthase TPS10 is not expressed in roots and does not display the haplotype-specific expression pattern. The results indicate that the same JA-mediated signaling cascade maycontrol the production of both the terpene (E)-β-caryophyllene and aromatic ester methyl anthranilate.