Prediction of protein loop geometries in solution

The ability to determine the structure of a protein in solution is a critical tool for structural biology, as proteins in their native state are found in aqueous environments. Using a physical chemistry based prediction protocol, we demonstrate the ability to reproduce protein loop geometries in experimentally derived solution structures. Predictions were run on loops drawn from (1)NMR entries in the Protein Databank (PDB), and from (2) the RECOORD database in which NMR entries from the PDB have been standardized and re-refined in explicit solvent. The predicted structures are validated by comparison with experimental distance restraints, a test of structural quality as defined by the WHAT IF structure validation program, root mean square deviation (RMSD) of the predicted loops to the original structural models, and comparison of precision of the original and predicted ensembles. Results show that for the RECOORD ensembles, the predicted loops are consistent with an average of 95%, 91%, and 87% of experimental restraints for the short, medium and long loops respectively. Prediction accuracy is strongly affected by the quality of the original models, with increases in the percentage of experimental restraints violated of 2% for the short loops, and 9% for both the medium and long loops in the PDB derived ensembles. We anticipate the application of our protocol to theoretical modeling of protein structures, such as fold recognition methods; as well as to experimental determination of protein structures, or segments, for which only sparse NMR restraint data is available.     

α1-AMP-Activated Protein Kinase Regulates Hypoxia-Induced Na,K-ATPase Endocytosis via Direct Phosphorylation of Protein Kinase Cζ

Hypoxia promotes Na,K-ATPase endocytosis via protein kinase Cζ (PKCζ)-mediated phosphorylation of the Na,K-ATPase α subunit. Here, we report that hypoxia leads to the phosphorylation of 5′-AMP-activated protein kinase (AMPK) at Thr172 in rat alveolar epithelial cells. The overexpression of a dominant-negative AMPK α subunit (AMPK-DN) construct prevented the hypoxia-induced endocytosis of Na,K-ATPase. The overexpression of the reactive oxygen species (ROS) scavenger catalase prevented hypoxia-induced AMPK activation. Moreover, hypoxia failed to activate AMPK in mitochondrion-deficient ρ⁰-A549 cells, suggesting that mitochondrial ROS play an essential role in hypoxia-induced AMPK activation. Hypoxia-induced PKCζ translocation to the plasma membrane and phosphorylation at Thr410 were prevented by the pharmacological inhibition of AMPK or by the overexpression of the AMPK-DN construct. We found that AMPK α phosphorylates PKCζ on residue Thr410 within the PKCζ activation loop. Importantly, the activation of AMPK α was necessary for hypoxia-induced AMPK-PKCζ binding in alveolar epithelial cells. The overexpression of T410A mutant PKCζ prevented hypoxia-induced Na,K-ATPase endocytosis, confirming that PKCζ Thr410 phosphorylation is essential for this process. PKCζ activation by AMPK is isoform specific, as small interfering RNA targeting the α1 but not the α2 catalytic subunit prevented PKCζ activation. Accordingly, we provide the first evidence that hypoxia-generated mitochondrial ROS lead to the activation of the AMPK α1 isoform, which binds and directly phosphorylates PKCζ at Thr410, thereby promoting Na,K-ATPase endocytosis.When exposed to low oxygen levels (hypoxia), cells develop adaptative strategies to maintain adequate levels of ATP (21). These strategies include increasing the efficiency of energy-producing pathways, mostly through anaerobic glycolysis, while decreasing energy-consuming processes such as Na,K-ATPase activity (30). Alveolar hypoxia occurs in many respiratory disorders, and it has been shown to decrease epithelial active Na⁺ transport, leading to impaired fluid reabsorption (37, 41, 42). Active Na⁺ transport and, thus, alveolar fluid reabsortion are effected mostly via apical sodium channels and the basolateral Na,K-ATPase (32, 38, 42). We have reported previously that hypoxia inhibits Na,K-ATPase activity by promoting its endocytosis from the plasma membrane by a mechanism that requires the generation of mitochondrial reactive oxygen species (ROS) and the phosphorylation of the Na,K-ATPase α subunit at Ser18 by protein kinase Cζ (PKCζ) (8, 9).The 5′-AMP-activated protein kinase (AMPK) is a heterotrimeric Ser/Thr kinase composed of a catalytic α subunit and regulatory β and γ subunits. Both isoforms of the AMPK catalytic subunit (α1 and α2) form complexes with noncatalytic subunits. The α1 subunit is ubiquitously expressed, whereas the α2 subunit isoform is expressed predominantly in tissues like the liver, heart, and skeletal muscle (36). The α1 and α2 subunit isoforms have ∼90% homology in their N-terminal catalytic domains and ∼60% homology in their C-terminal domains (36), suggesting that they may have distinct downstream targets (31). AMPK activation requires phosphorylation at Thr172 in the activation loop of the α subunit by upstream kinases (12, 19). Findings from recent studies suggest that AMPK is an important signaling intermediary in coupling ion transport and metabolism (15). Indeed, it has been reported that the pharmacological activation of AMPK inhibits amiloride- and ouabain-sensitive epithelial Na⁺ transport (15). Moreover, the activities of the epithelial Na⁺ channel (ENaC) (2, 17), the Na,K-ATPase (40), and the cystic fibrosis transmembrane conductance regulator (17) have been shown to be inhibited by AMPK. Here, we provide evidence that hypoxia, via mitochondrial ROS, leads to AMPK activation and that AMPK binds to and directly phosphorylates PKCζ in an isoform-specific manner, thus promoting Na,K-ATPase endocytosis in alveolar epithelial cells (AEC).     

The dorsal neural tube: A dynamic setting for cell fate decisions

The dorsal neural tube first generates neural crest cells that exit the neural primordium following an epithelial‐to‐mesenchymal conversion to become sympathetic ganglia, Schwann cells, dorsal root sensory ganglia, and melanocytes of the skin. Following the end of crest emigration, the dorsal midline of the neural tube becomes the roof plate, a signaling center for the organization of dorsal neuronal cell types. Recent lineage analysis performed before the onset of crest delamination revealed that the dorsal tube is a highly dynamic region sequentially traversed by fate‐restricted crest progenitors. Furthermore, prospective roof plate cells were shown to originate ventral to presumptive crest and to progressively relocate dorsalward to occupy their definitive midline position following crest delamination. These data raise important questions regarding the mechanisms of cell emigration in relation to fate acquisition, and suggest the possibility that spatial and/or temporal information in the dorsal neural tube determines initial segregation of neural crest cells into their derivatives. In addition, they emphasize the need to address what controls the end of neural crest production and consequent roof plate formation, a fundamental issue for understanding the separation between central and peripheral lineages during development of the nervous system. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 796–812, 2010.     

F-Spondin, expressed in somite regions avoided by neural crest cells, mediates inhibition of distinct somite domains to neural crest migration

Neural crest (NC) cells migrate exclusively into the rostral half of each sclerotome, where they avoid the dermomyotome and the paranotochordal sclerotome. F-spondin is expressed in these inhibitory regions and throughout the caudal halves. In vitro bioassays of NC spreading on substrates of rostral or caudal epithelial-half somites (RS or CS, respectively) revealed that NC cells adopt on RS a fibroblastic morphology, whereas on CS they fail to flatten. F-spondin inhibited flattening of NC cells on RS. Conversely, F-spondin antibodies prevented rounding up of NC cells on CS. Addition of F-spondin to trunk explants inhibited NC migration into the sclerotome, and treatment of embryos with anti-F-spondin antibodies yielded migration into otherwise inhibitory sites. Thus, somite-derived F-spondin is an inhibitory signal involved in patterning the segmental migration of NC cells and their topographical segregation within the RS.     

Analysis of chloroquine resistance transporter (CRT) isoforms and orthologues in S. cerevisiae yeast

Previous work from our laboratory optimized MeOH-inducible expression of the P. falciparum malarial parasite transporter PfCRT in P. pastoris yeast. These strains are useful for many experiments but do not allow for inducible protein expression under ambient growth conditions. We have therefore optimized galactose-inducible expression of PfCRT in S. cerevisiae yeast. We find that expression of PfCRT confers CQ hypersensitivity to growing yeast and that this is due to plasma membrane localization of the transporter. We use quantitative analyses of growth rates to compare hypersensitivity for yeast expressing various PfCRT isoforms. We also report successful high level inducible expression of the P. vivax orthologue, PvCRT, and compare CQ hypersensitivity for PvCRT vs PfCRT expressing yeast. We test the hypothesis that hypersensitivity is due to increased transport of CQ into yeast expressing the transporters via direct (3)H-CQ transport experiments and analyze the effect that membrane potential has on transport. The data suggest important new tools for rapid functional screening of PfCRT and PvCRT isoforms and provide further evidence for a model wherein membrane potential promotes charged CQ transport by PfCRT. Data also support our previous conclusion that wild type PfCRT is capable of CQ transport and provide a basis for understanding the lack of correspondence between PvCRT mutations and resistance to CQ in the important malarial parasite P. vivax.     

Matrix proteins are inefficiently imported into Arabidopsis peroxisomes lacking the receptor-docking peroxin PEX14

Mutations in peroxisome biogenesis proteins (peroxins) can lead to developmental deficiencies in various eukaryotes. PEX14 and PEX13 are peroxins involved in docking cargo-receptor complexes at the peroxisomal membrane, thus aiding in the transport of the cargo into the peroxisomal matrix. Genetic screens have revealed numerous Arabidopsis thaliana peroxins acting in peroxisomal matrix protein import; the viable alleles isolated through these screens are generally partial loss-of-function alleles, whereas null mutations that disrupt delivery of matrix proteins to peroxisomes can confer embryonic lethality. In this study, we used forward and reverse genetics in Arabidopsis to isolate four pex14 alleles. We found that all four alleles conferred reduced PEX14 mRNA levels and displayed physiological and molecular defects suggesting reduced but not abolished peroxisomal matrix protein import. The least severe pex14 allele, pex14-3, accumulated low levels of a C-terminally truncated PEX14 product that retained partial function. Surprisingly, even the severe pex14-2 allele, which lacked detectable PEX14 mRNA and PEX14 protein, was viable, fertile, and displayed residual peroxisome matrix protein import. As pex14 plants matured, import improved. Together, our data indicate that PEX14 facilitates, but is not essential for peroxisomal matrix protein import in plants.     

Characterization of T-lymphocyte subpopulations infiltrating primary breast cancer

Summary Characterization of T-lymphocyte subpopulations adjacent to and infiltrating the primary tumor of breast cancer was carried out using a direct immunofluorescence procedure with the antibodies anti-(Leu-2a) for suppressor/cytotoxic (CD8⁺) and anti-(Leu-3a) for helper/inducer (CD4⁺) T-lymphocytes. Fifty-six primary malignant tumors with lymphoid infiltration were studied. The majority (58.9%) were infiltrating duct carcinoma. There were metastases to axillary lymph nodes in 6.67% of the patients. Massive lymphoid infiltration (>40 lymphocytes per ×400 microscopic field) was found in 19.6% of the tumors and moderate infiltration (20–40 lymphocytes per field) in 51.8%. In all the tumors studied there was a reversed CD4⁺/CD8⁺ ratio as compared to that found in normal peripheral blood. In 66.1% the CD4⁺/CD8⁺ ratio (helper/suppressor) was less than 1.0. The reversed ratio was due to a significant decrease in the number of helper cells (P<0.0005). The most significant drop was in the stroma area (P<0.0001) as well as in the tumor tissue (P=0.001). Of particular interest was the significant positive correlation between the age of the patients and an increased number of CD4⁺lymphocytes in the stroma (P=0.02). Significant negative correlations were found between a reduced number of CD4⁺ lymphocytes or CD4⁺/CD8⁺ ratio and several histological parameters: tumor diameter, pleomorphism, nucleus/cytoplasm ratio. There was also a significant positive correlation between the total number of CD8⁺ lymphocytes infiltrating the tumor tissue and the number of axillary lymph nodes with metastatic disease (P=0.03). It is suggested that the reversed ratio of CD4⁺/CD8⁺ lymphocytes may significantly affect the host/tumor immune surveillance.     

Calnexin overexpression sensitizes recombinant CHO cells to apoptosis induced by sodium butyrate treatment

Sodium butyrate (NaBu) can enhance the expression of foreign genes in recombinant Chinese hamster ovary (rCHO) cells, but it can also inhibit cell growth and induce cellular apoptosis. In this study, the potential role of calnexin (Cnx) expression in rCHO cells treated with 5 mM NaBu was investigated for rCHO cells producing tumor necrosis factor receptor FC. To regulate the Cnx expression level, a tetracycline-inducible system was used. Clones with different Cnx expression levels were selected and investigated. With regard to productivity per cell (q_p), NaBu enhanced the q_p by over twofold. Under NaBu treatment, Cnx overexpression further enhanced the q_p by about 1.7-fold. However, under NaBu stress, the cells overexpressing Cnx showed a poorer viability profile with a consistent difference of over 25% in the viability when compared to the Cnx-repressed condition. This drop in the viability was attributed to increased apoptosis seen in these cells as evidenced by enhanced poly (ADP-ribose) polymerase cleavage and cytochrome C release. Ca²⁺ localization staining and subsequent confocal imaging revealed elevated cytosolic Ca²⁺ ([Ca²⁺]_c) in the Cnx-overexpressing cells when compared to the Cnx-repressed condition, thus endorsing the increased apoptosis observed in these cells. Taken together, Cnx overexpression not only improved the q_p of cells treated with NaBu, but it also sensitized cells to apoptosis.     

pH-dependent interconversion of two forms of tyrosinase in human skin.

1. We have shown that the characteristic lag in cresolase activity of human skin tyrosinase at inhibitory concentration of tyrosine was absent at all pH values studied, i.e. pH 5.2, 5.7, 6.2 and 6.8, if the enzyme solubilized at low pH was used as the source of enzyme, but the same enzyme when dialysed against buffers of various pH values showed linear activity only at pH 5.2 and was not inhibited by excess tyrosine, whereas at higher pH values it exhibited a lag and inhibition by excess tyrosine. 2. However, the enzyme solubilized in buffer/detergent, pH 6.8, when dialysed against buffer of the same pH showed linear activity at pH 5.2 and non-linear activity at pH 6.8. 3. The water/detergent-solubilized enzyme from human skin melanosomes showed linear activity even at inhibitory concentrations of tyrosine at pH 5.2 and 6.8 up to 2 h, but acceleration of rate was observed after 2 h for the enzyme measured at pH 6.8. 4. After dialysis of the water/detergent-solubilized enzyme against double-glass-distilled water, it still exhibits linear activity at inhibitory concentration of tyrosines at pH 6.8 for the first 2 h, but the same enzyme when dialysed against 0.02 M-sodium phosphate buffer, pH 6.8, exhibits negligible activity up to 1/2 h, in contrast with considerable activity before dialysis during the same interval of time, but without any loss of activity at later intervals of incubation time. 5. On the basis of these results, it is concluded that the enzyme exists in at least two interconvertible forms, one without lag and inhibition by excess tyrosine and the other with lag and inhibition by excess tyrosine. These two forms are interconvertible only by gradual change in pH over a period of hours.     

Staurosporine Induces Astrocytic Phenotypes and Differential Expression of Specific PKC Isoforms in C6 Glial Cells

Abstract: In this study we examined the effects of staurosporine, a potent inhibitor of protein kinase C (PKC), on the differentiation of C6 glial cells and on the expression and cellular distribution of specific PKC isoforms. Staurosporine reduced cell proliferation and induced distinctive changes in the morphological appearance of the cells to that characteristic of cells exhibiting astrocytic phenotypes. The differentiative effect of staurosporine was further indicated by the increased expression of two proteins related to astrocytic phenotypes, glial fibrillary acidic protein (GFAP) and glutamine synthetase. Thus, staurosporine induced a dose-dependent increase both in GFAP immunoreactivity and in the activity and protein levels of glutamine synthetase. Staurosporine also induced a decrease in the expression of PKC-β₂ and an increase in that of PKC-γ. In addition, it induced translocation of PKC-ε from the membrane to the cytosol, whereas no differences were observed in the distribution of the other PKC isoforms. The results of our study indicate that staurosporine induced astrocytic phenotypes in glial cells and that changes in the expression and cellular distribution of these PKC isoforms may be related to astrocytic differentiation.