Density Functional study of the photoactive yellow protein's chromophore

We have investigated the structural and electronic properties of p-coumaric acid, the chromophore of the photoactive yellow protein (PYP), by means of first-principles molecular dynamics based on density functional theory (DFT). We have studied the chromophore both in the vacuum and in an extended model which includes the nearest residues in the binding pocket of PYP, as derived from crystallographic data. We have characterized the ground state of the isolated chromophore in its protonated and deprotonated forms and computed the energy barrier involved in the trans to cis isomerization process around the carbon-carbon double bond. A comparison of the optimized structures of the chromophore in the vacuum and in the extended protein model, both in the trans (ground state of PYP in the dark) and cis (first light-activated intermediate) configuration, shows how the protein environment affects the chromophore in the first step of the photocycle. Our model gives an energy storage of 25 kcal/mol associated with the trans-to-cia photoisomerization. Finally, we have elucidated the nature of the electronic excitation relevant for the photochemistry of PYP by means of time-dependent DFT calculations.

Galectin-9 protein expression in endothelial cells is positively regulated by histone deacetylase 3

Galectin-9 expression in endothelial cells can be induced in response to inflammation. However, the mechanism of its expression remains unclear. In this study, we found that interferon-? (IFN-?) induced galectin-9 expression in human endothelial cells in a time-dependent manner, which coincided with the activation of histone deacetylase (HDAC). When endothelial cells were treated with the HDAC3 inhibitor, apicidin, or shRNA-HDAC3 knockdown, IFN-?-induced galectin-9 expression was abolished. Overexpression of HDAC3 induced the interaction between phosphoinositol 3-kinase (PI3K) and IFN response factor 3 (IRF3), leading to IRF3 phosphorylation, nuclear translocation, and galectin-9 expression. HDAC3 functioned as a scaffold protein for PI3K/IRF3 interaction. In addition to galectin-9 expression, IFN-? also induced galectin-9 location onto plasma membrane, which was HDAC3-independent. Importantly, HDAC3 was essential for the constitutive transcription of PI3K and IRF3, which might be responsible for the basal level of galectin-9 expression. The phosphorylation of IRF3 was essential for galectin-9 expression. This study provides new evidence that HDAC3 regulates galectin-9 expression in endothelial cells via interaction with PI3K-IRF3 signal pathway.

Klebsiella pneumoniae outer membrane protein A is required to prevent the activation of airway epithelial cells

Outer membrane protein A (OmpA) is a class of proteins highly conserved among the Enterobacteriaceae family and throughout evolution. Klebsiella pneumoniae is a capsulated Gram-negative pathogen. It is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by a lack of an early inflammatory response. Data from our laboratory indicate that K. pneumoniae CPS helps to suppress the host inflammatory response. However, it is unknown whether K. pneumoniae employs additional factors to modulate host inflammatory responses. Here, we report that K. pneumoniae OmpA is important for immune evasion in vitro and in vivo. Infection of A549 and normal human bronchial cells with 52OmpA2, an ompA mutant, increased the levels of IL-8. 52145-?wca ompA, which does not express CPS and ompA, induced the highest levels of IL-8. Both mutants could be complemented. In vivo, 52OmpA2 induced higher levels of tnfa, kc, and il6 than the wild type. ompA mutants activated NF-?B, and the phosphorylation of p38, p44/42, and JNK MAPKs and IL-8 induction was via NF-?B-dependent and p38- and p44/42-dependent pathways. 52OmpA2 engaged TLR2 and -4 to activate NF-?B, whereas 52145-?wca ompA activated not only TLR2 and TLR4 but also NOD1. Finally, we demonstrate that the ompA mutant is attenuated in the pneumonia mouse model. The results of this study indicate that K. pneumoniae OmpA contributes to attenuate airway cell responses. This may facilitate pathogen survival in the hostile environment of the lung. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Complement factor H is a serum-binding protein for adrenomedullin, and the resulting complex modulates the bioactivities of both partners

Adrenomedullin (AM) is an important regulatory peptide involved in both physiological and pathological states. We have previously demonstrated the existence of a specific AM-binding protein (AMBP-1) in human plasma. In the present study, we developed a nonradioactive ligand blotting assay, which, together with high pressure liquid chromatography/SDS-polyacrylamide gel electrophoresis purification techniques, allowed us to isolate AMBP-1 to homogeneity. The purified protein was identified as human complement factor H. We show that AM/factor H interaction interferes with the established methodology for quantification of circulating AM. Our data suggest that this routine procedure does not take into account the AM bound to its binding protein. In addition, we show that factor H affects AM in vitro functions. It enhances AM-mediated induction of cAMP in fibroblasts, augments the AM-mediated growth of a cancer cell line, and suppresses the bactericidal capability of AM on Escherichia coli. Reciprocally, AM influences the complement regulatory function of factor H by enhancing the cleavage of C3b via factor I. In summary, we report on a potentially new regulatory mechanism of AM biology, the influence of factor H on radioimmunoassay quantification of AM, and the possible involvement of AM as a regulator of the complement cascade.

Comparison of dynamics of wildtype and V94M human UDP-galactose 4-epimerase-A computational perspective on severe epimerase-deficiency galactosemia

UDP-galactose 4'-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr157) and the enzyme-bound NAD(+) cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (kcat), with less substantial effects on Km. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia.

Apolipoprotein C-III protein concentrations and gene polymorphisms in type 1 diabetes:associations with lipoprotein subclasses

Serum apolipoprotein C-III (apoCIII) concentration and apoCIII gene polymorphisms have been shown to be a risk factor for cardiovascular disease; however, the underlying mechanisms remain unclear. In addition, no studies have been performed that address these issues in type 1 diabetes. The current study investigated apoCIII protein and apoCIII gene variation in a normotriglyceridemic (82 +/- 57 mg/dL) population of patients with type 1 diabetes, the Diabetes Control and Complications Trial/Epidemiology of Diabetes Intervention and Complications (DCCT/EDIC) cohort. Blood samples were obtained in 409 patients after an overnight fast. Serum apoCIII concentration was highly correlated with multiple changes in lipids and lipoproteins that resulted in an adverse cardiovascular disease risk profile. Higher apoCIII concentrations were associated (P <.0001) with increased triglycerides (r = 0.78), total (r = 0.61) and low-density lipoprotein (LDL) (r = 0.40) cholesterol, apoA-I (r = 0.26), and apoB (r = 0.50), and these relationships persisted after controlling for age, gender, body mass index (BMI), and hemoglobin A1c (HbA1c). Nuclear magnetic resonance (NMR) lipoprotein subclass analyses demonstrated that apoCIII was correlated with an increase in very-low-density lipoprotein (VLDL) subclasses (P = .0001). There also was a highly significant positive relationship between serum apoCIII concentration and the LDL particle concentration in both men (r = 0.49, P = .001) and women (r = 0.40, P = .001), and a highly significant negative relationship between serum apoCIII levels and average LDL particle size in both men (r = -0.37, P = .001) and women (r = -0.22, P = .001) due primarily to an augmentation in the small L1 subclass (r = 0.42, P = .0001). Neither the T(-455) --> C polymorphism affecting an insulin response element in the apoCIII gene promoter nor a SacI polymorphism in the 3'UTR were associated with any alterations in circulating apoCIII concentrations, serum lipids, apolipoprotein concentrations, lipoprotein composition, or parameters measured by NMR lipoprotein subclass analyses. In summary, elevated apoCIII concentration was associated with risk factors for cardiovascular disease in normolipidemic type 1 diabetic patients through associated changes in lipoprotein subfraction distributions, which were independent of apoCIII genotype.

Metabolism of very low- and low-density lipoproteins isolated from normolipidaemic type 2 (non-insulin-dependent) diabetic patients by human monocyte-derived macrophages

The very low- and low-density lipoprotein fractions were isolated from 16 normolipidaemic Type 2 (non-insulin-dependent) diabetic patients in good to fair glycaemic control and from corresponding age-, sex-, and race-matched, non-diabetic control subjects. Rates of cholesteryl ester synthesis averaged 268 +/- 31 vs 289 +/- 40 pmol 14C-cholesteryl oleate.mg cell protein-1.20 h-1 for very low- and 506 +/- 34 vs 556 +/- 51 pmol 14C-cholesteryl oleate.mg cell protein-1.20 h-1 for low-density lipoproteins isolated from the Type 2 diabetic patients and control subjects, respectively, when they were incubated with human macrophages. A group of approximately one-third of the patients was selected for separate analyses because very low-density lipoproteins isolated from these patients did stimulate more cholesteryl ester synthesis when incubated with macrophages. There were no significant differences in the lipid composition of the lipoproteins isolated from the three groups of subjects. The relative proportion of apoprotein C to apoprotein E was significantly decreased (p less than 0.002) in the very low-density lipoproteins from diabetic patients and was further decreased in samples from these selected diabetic patients. The apoprotein C-I content of very low-density lipoproteins isolated from diabetic patients was increased compared to control subjects and was further increased in samples from the selected diabetic patients (p less than 0.02). There were no significant differences in the proportions of apoproteins C-III-0, C-III-1, or C-III-2 among the three groups. These studies suggest that in normolipidaemic Type 2 diabetic patients, the apoprotein composition of VLDL is abnormal and this may alter VLDL macrophage interactions and thus contribute to the increased prevalence of atherosclerosis in diabetic patients.

Fructose Chemistry

Fructose is a six-carbon ketose monosaccharide. In aqueous solution and in the crystalline form, the majority of the molecules form ring structures. Of these, the six-membered pyranose form is the most abundant; however, about one-quarter of the molecules are in the five-membered, furanose form. While many of its reactions are similar to those of glucose, the presence of a ketone group in the chain, and the relative ease with which the molecule forms a five-membered furanose ring affects its chemistry and biochemistry. Specific pathways are required to enable organisms to exploit fructose in energy metabolism; these require the enzyme fructokinase and involve the conversion of fructose to glycolytic intermediates. Similarly, specific pathways for the biosynthesis of fructose and fructose-containing polymers, such as inulin, are required. Non-enzymatic glycation (fructation) by fructose has not been as extensively studied as the corresponding reactions with glucose. Nevertheless, especially in diabetic patients and fructose-rich foodstuffs, this reaction is likely to be important.

Protein destabilisation by ruthenium(II) tris-bipyridine based protein-surface mimetics

Highly functionalised ruthenium(II) tris-bipyridine receptor 1 which acts as a selective sensor for equine cytochrome c (cyt c) is shown to destabilise the native protein conformation by around 25 degrees C. Receptors 2 and 3 do not exert this effect confirming the behaviour is a specific effect of molecular recognition between 1 and cyt c, whilst the absence of a destabilising effect on 60% acetylated cyt c demonstrates the behaviour of 1 to be protein specific. Molecular recognition also modifies the conformational properties of the target protein at room temperature as evidenced by ion-mobility spectrometry (IMS) and accelerated trypsin proteolysis.

Proteomics on porcine haptoglobin and IgG/IgA show protein species distribution and glycosylation pattern to remain similar in PCV2-SD infection

Haptoglobin (Hp) and immunoglobulins are plasma glycoproteins involved in the immune reaction of the organism after infection and/or inflammation. Porcine circovirus type 2-systemic disease (PCV2-SD), formerly known as postweaning multisystemic wasting syndrome (PMWS), is a globally spread pig disease of great economic impact. PCV2-SD affects the immunological system of pigs causing immunosuppression. The aim of this work was to characterize the Hp protein species of healthy and PCV2-SD affected pigs, as well as the protein backbone and the glycan chain composition of porcine Hp. PCV2-SD affected pigs had an increased overall Hp level, but it did not affect the ratio between Hp species. Glycoproteomic analysis of the Hp β subunits confirmed that porcine Hp is N-glycosylated and, unexpectedly, O-glycosylated, a PTM that is not found on Hp from healthy humans. The glyco-profile of porcine IgG and IgA heavy chains was also characterized; decreased levels of both proteins were found in the investigated group of PCV2-SD affected pigs. Obtained results indicate that no significant changes in the N- and O-glycosylation patterns of these major porcine plasma glycoproteins were detectable between healthy and PCV2-SD affected animals.

Unspliced X-box-binding Protein 1 (XBP1) Protects Endothelial Cells from Oxidative Stress through Interaction with Histone Deacetylase 3

It is well-known that atherosclerosis occurs geographically at branch points where disturbed flow predisposes to the development of plaque via triggering of oxidative stress and inflammatory reactions. In this study, we found that disturbed flow activated anti-oxidative reactions via up-regulating heme oxygenase 1 (HO-1) in an X-box binding protein 1 (XBP1) and histone deacetylase 3 (HDAC3)-dependent manner. Disturbed flow concomitantly up-regulated the unspliced XBP1 (XBP1u) and HDAC3 in a vascular endothelial growth factor receptor (VEGFR) and PI3K/Akt dependent manner. The presence of XBP1 was essential for the up-regulation of HDAC3 protein. Over-expression of XBP1u and/or HDAC3 activated Akt1 phosphorylation, Nrf2 protein stabilization and nuclear translocation, and HO-1 expression. Knockdown of XBP1u decreased the basal level and disturbed flow-induced Akt1 phosphorylation, Nrf2 stabilization and HO-1 expression. Knockdown of HDAC3 ablated XBP1u-mediated effects. The mammalian target of rapamycin complex 2 (mTORC2) inhibitor, AZD2014, ablated XBP1u or HDAC3 or disturbed flow-mediated Akt1 phosphorylation, Nrf2 nuclear translocation and HO-1 expression. Neither actinomycin D nor cycloheximide affected disturbed flow-induced up-regulation of Nrf2 Protein. Knockdown of Nrf2 abolished XBP1u or HDAC3 or disturbed flow-induced HO-1 up-regulation. Co-immunoprecipitation assays demonstrated that XBP1u physically bound to HDAC3 and Akt1. The region of amino acids 201 to 323 of the HDAC3 protein was responsible for the binding to XBP1u. Double immunofluorescence staining revealed that the interactions between Akt1 and mTORC2, Akt1 and HDAC3, Akt1 and XBP1u, HDAC3 and XBP1u occurred in the cytosol. Thus, we demonstrate that XBP1u and HDAC3 exert a protective effect on disturbed flow-induced oxidative stress via up-regulation of mTORC2-dependent Akt1 phosphorylation and Nrf2-mediated HO-1 expression.

Resonantly Enhanced Multi-Photon Ionization Spectrum of the Neutral Green Fluorescent Protein Chromophore

The photophysics of the green fluorescent protein is governed by the electronic structure of the chromophore at the heart of its β-barrel protein structure. We present the first two-color, resonance-enhanced, multiphoton ionization spectrum of the isolated neutral chromophore in vacuo with supporting electronic structure calculations. We find the absorption maximum to be 3.65 ± 0.05 eV (340 ± 5 nm), which is blue-shifted by 0.5 eV (55 nm) from the absorption maximum of the protein in its neutral form. Our results show that interactions between the chromophore and the protein have a significant influence on the electronic structure of the neutral chromophore during photoabsorption and provide a benchmark for the rational design of novel chromophores as fluorescent markers or photomanipulators.

CCAAT/enhancer binding protein α predicts poorer prognosis and prevents energy starvation-induced cell death in hepatocellular carcinoma

CCAAT enhancer binding protein α (C/EBPα) plays an essential role in cellular differentiation, growth, and energy metabolism. Here, we investigate the correlation between C/EBPα and hepatocellular carcinoma (HCC) patient outcomes and how C/EBPα protects cells against energy starvation. Expression of C/EBPα protein was increased in the majority of HCCs examined (191 pairs) compared with adjacent nontumor liver tissues in HCC tissue microarrays. Its upregulation was correlated significantly with poorer overall patient survival in both Kaplan-Meier survival (P = 0.017) and multivariate Cox regression (P = 0.028) analyses. Stable C/EBPα-silenced cells failed to establish xenograft tumors in nude mice due to extensive necrosis, consistent with increased necrosis in human C/EBPα-deficient HCC nodules. Expression of C/EBPα protected HCC cells in vitro from glucose and glutamine starvation-induced cell death through autophagy-involved lipid catabolism. Firstly, C/EBPα promoted lipid catabolism during starvation, while inhibition of fatty acid beta-oxidation significantly sensitized cell death. Secondly, autophagy was activated in C/EBPα-expressing cells, and the inhibition of autophagy by ATG7 knockdown or chloroquine treatment attenuated lipid catabolism and subsequently sensitized cell death. Finally, we identified TMEM166 as a key player in C/EBPα-mediated autophagy induction and protection against starvation.

CONCLUSION: The C/EBPα gene is important in that it links HCC carcinogenesis to autophagy-mediated lipid metabolism and resistance to energy starvation; its expression in HCC predicts poorer patient prognosis.

Proteomic identification of plasma proteins as markers of growth promoter abuse in cattle

Growth-promoting agents are continually misused for increasing animal growth and fraudulent gain in the meat industry, yet detection rates from conventional targeted testing for drug residues do not reflect this. This is because testing currently relies on direct detection of drugs or related metabolites and administrators of such compounds can take adaptive measures to avoid detection through the use of endogenous or unknown drugs, and low dose or combined mixtures. New detection methods are needed which focus on the screening of biological responses of an animal to such growth-promoting agents as it has been demonstrated that genomic, proteomic and metabolomics profiles are altered by xenobiotic intake. Therefore, an untargeted proteomics approach using comparative two-dimensional gel electrophoresis (2DE) was carried out to identify putative proteins altered in plasma after treatment with oestradiol, dexamethasone or prednisolone. Twenty-four male cattle were randomly assigned to four groups (n = 6) for experimental treatment over 40 days, namely a control group of non-treated cattle, and three groups administered 17β-oestradiol-3-benzoate (0.01 mg/kg, intramuscular), dexamethasone sodium phosphate (0.7 mg/day, per os) or prednisolone acetate (15 mg/day, per os), respectively. Plasma collected from each animal at day 25 post study initiation was subjected to proteomic analysis by 2DE for comparison of protein expression between treated and untreated animals. Analysis of acquired gel images revealed 22 plasma proteins which differed in expression by more than 50 % (p < 0.05) in treated animals compared to untreated animals. Proteins of interest underwent identification by LC–MS/MS analysis and were found to have associated roles in transport, blood coagulation, immune response and metabolism pathways. In this way, seven proteins are highlighted as novel biomarker candidates including transthyretin which is shown to be significantly increased in all treatment groups compared to control animals and potentially may find use as global markers of suspect anabolic practice.