Heat shock protein 27 controls apoptosis by regulating Akt activation

Activation of the serine-threonine kinase Akt by cytokines, chemokines, and bacterial products delays constitutive neutrophil apoptosis, resulting in a prolonged inflammatory response. We showed previously that Akt exists in a signaling complex with p38 MAPK, MAPK-activated protein kinase-2 (MAPKAPK-2), and heat shock protein-27 (Hsp27); and Hsp27 dissociates from the complex upon neutrophil activation. To better understand the regulation of this signaling module, the hypothesis that Akt phosphorylation of Hsp27 regulates its interaction with Akt was tested. The present study shows that Akt phosphorylated Hsp27 on Ser-82 in vitro and in intact cells, and phosphorylation of Hsp27 resulted in its dissociation from Akt. Additionally, the interaction between Hsp27 and Akt was necessary for activation of Akt in intact neutrophils. Constitutive neutrophil apoptosis was accelerated by sequestration of Hsp27 from Akt, and this enhanced rate of apoptosis was reversed by introduction of constitutively active recombinant Akt. Our results define a new mechanism by which Hsp27 regulates apoptosis, through control of Akt activity.     

AGenDA: homology-based gene prediction

We present a www server for homology-based gene prediction. The user enters a pair of evolutionary related genomic sequences, for example from human and mouse. Our software system uses CHAOS and DIALIGN to calculate an alignment of the input sequences and then searches for conserved splicing signals and start/stop codons around regions of local sequence similarity. This way, candidate exons are identified that are used, in turn, to calculate optimal gene models. The server returns the constructed gene model by email, together with a graphical representation of the underlying genomic alignment.     

Functional expression and direct visualization of the human alpha 2B -adrenergic receptor and alpha 2B -AR-green fluorescent fusion protein in mammalian cell using Semliki Forest virus vectors

The alpha 2B -adrenergic receptor ( alpha 2B -AR), a member of the G protein-coupled receptor (GPCR) superfamily, was expressed at high levels from Semliki Forest virus (SFV) vectors in mammalian cells. Constructs were engineered by fusing enhanced green fluorescent protein (eGFP) and the SFV capsid to opposite ends of the alpha 2B -AR. The receptor fusions alpha 2B -AR-eGFP and CAP- alpha 2B -AR expressed in CHO-K1 cells generated alpha 2B values of 176 and 122pmol/mg of membrane protein, respectively, and showed similar ligand binding characteristics, alpha 2B -AR subtype-selectivity, and G protein activation as reported for stable expression in CHO-K1 cells. Cryo-electron microscopy and eGFP-based fluorescence indicated the same subcellular receptor distribution. SFV expression is well suited for studies on the pharmacology, biochemistry, and cell biology of GPCRs, and for large-scale recombinant protein production in mammalian suspension culture to generate sufficient receptor quantities for structural biology.     

Extrapolation of Inter Domain Communications and Substrate Binding Cavity of Camel HSP70 1A: A Molecular Modeling and Dynamics Simulation Study

Heat shock protein 70 (HSP70) is an important chaperone, involved in protein folding, refolding, translocation and complex remodeling reactions under normal as well as stress conditions. However, expression of HSPA1A gene in heat and cold stress conditions associates with other chaperons and perform its function. Experimental structure for Camel HSP70 protein (cHSP70) has not been reported so far. Hence, we constructed 3D models of cHSP70 through multi- template comparative modeling with HSP110 protein of S. cerevisiae (open state) and with HSP70 protein of E. coli 70kDa DnaK (close state) and relaxed them for 100 nanoseconds (ns) using all-atom Molecular Dynamics (MD) Simulation. Two stable conformations of cHSP70 with Substrate Binding Domain (SBD) in open and close states were obtained. The collective mode analysis of different transitions of open state to close state and vice versa was examined via Principal Component Analysis (PCA) and Minimum Distance Matrix (MDM). The results provide mechanistic representation of the communication between Nucleotide Binding Domain (NBD) and SBD to identify the role of sub domains in conformational change mechanism, which leads the chaperone cycle of cHSP70. Further, residues present in the chaperon functioning site were also identified through protein-peptide docking. This study provides an overall insight into the inter domain communication mechanism and identification of the chaperon binding cavity, which explains the underlying mechanism involved during heat and cold stress conditions in camel.     

Direct Determination of Phosphatase Activity from Physiological Substrates in Cells

A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP) performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min^-1 mg^-1 for PPi, to 56 ± 11 nmol min^-1 mg^-1 for AMP, to 79 ± 23 nmol min^-1 mg^-1 for beta-glycerophosphate and to 73 ± 15 nmol min^-1 mg^-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole – a TNAP inhibitor- served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC₅₀ value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.     

Upregulation of miR21 and Repression of Grhl3 by Leptin Mediates Sinusoidal Endothelial Injury in Experimental Nonalcoholic Steatohepatitis

Sinusoidal endothelial dysfunction (SED) has been found to be an early event in nonalcoholic steatohepatitis (NASH) progression but the molecular mechanisms underlying its causation remains elusive. We hypothesized that adipokine leptin worsens sinusoidal injury by decreasing functionally active nitric oxide synthase 3 (NOS)3 via miR21. Using rodent models of NASH, and transgenic mice lacking leptin and leptin receptor, results showed that hyperleptinemia caused a 4–5 fold upregulation of hepatic miR21 as assessed by qRTPCR. The upregulation of miR21 led to a time-dependent repression of its target protein Grhl3 levels as shown by western blot analyses. NOS3-p/NOS3 ratio which is controlled by Grhl3 was significantly decreased in NASH models. SED markers ICAM-1, VEGFR-2, and E-selectin as assessed by immunofluorescence microscopy were significantly up regulated in the progressive phases of NASH. Lack of leptin or its receptor in vivo, reversed the upregulation of miR21 and restored the levels of Grhl3 and NOS3-p/NOS3 ratio coupled with decreased SED dysfunction markers. Interestingly, leptin supplementation in mice lacking leptin, significantly enhanced miR21 levels, decreased Grhl3 repression and NOS3 phosphorylation. Leptin supplementation in isolated primary endothelial cells, Kupffer cells and stellate cells showed increased mir21 expression in stellate cells while sinusoidal injury was significantly higher in all cell types. Finally miR21 KO mice showed increased NOS3-p/NOS3 ratio and reversed SED markers in the rodent models of NASH. The experimental results described here show a close association of leptin-induced miR21 in aiding sinusoidal injury in NASH.     

LucY: A Versatile New Fluorescent Reporter Protein

We report on the discovery, isolation, and use of a novel yellow fluorescent protein. Lucigen Yellow (LucY) binds one FAD molecule within its core, thus shielding it from water and maintaining its structure so that fluorescence is 10-fold higher than freely soluble FAD. LucY displays excitation and emission spectra characteristic of FAD, with 3 excitation peaks at 276nm, 377nm, and 460nm and a single emission peak at 530nm. These excitation and emission maxima provide the large Stokes shift beneficial to fluorescence experimentation. LucY belongs to the MurB family of UDP-N-acetylenolpyruvylglucosamine reductases. The high resolution crystal structure shows that in contrast to other structurally resolved MurB enzymes, LucY does not contain a potentially quenching aromatic residue near the FAD isoalloxazine ring, which may explain its increased fluorescence over related proteins. Using E. coli as a system in which to develop LucY as a reporter, we show that it is amenable to circular permutation and use as a reporter of protein-protein interaction. Fragmentation between its distinct domains renders LucY non-fluorescent, but fluorescence can be partially restored by fusion of the fragments to interacting protein domains. Thus, LucY may find application in Protein-fragment Complementation Assays for evaluating protein-protein interactions.     

Endothelial Nitric Oxide Synthase Deficient Mice Are Protected from Lipopolysaccharide Induced Acute Lung Injury

Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS^-/-) are protected against ALI. In both wild-type and eNOS^-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS^-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS^-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS^-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS^-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr³⁴. Finally, we found that the reduction in NOS uncoupling in eNOS^-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.