Crystal structure of Arabidopsis cyclophilin38 reveals a previously uncharacterized immunophilin fold and a possible autoinhibitory mechanism

Cyclophilin38 (CYP38) is one of the highly divergent cyclophilins from Arabidopsis thaliana. Here, we report the crystal structure of the At-CYP38 protein (residues 83 to 437 of 437 amino acids) at 2.39-Å resolution. The structure reveals two distinct domains: an N-terminal helical bundle and a C-terminal cyclophilin β-barrel, connected by an acidic loop. Two N-terminal β-strands become part of the C-terminal cyclophilin β-barrel, thereby making a previously undiscovered domain organization. This study shows that CYP38 does not possess peptidyl-prolyl cis/trans isomerase activity and identifies a possible interaction of CYP38 with the E-loop of chlorophyll protein47 (CP47), a component of photosystem II. The interaction of CYP38 with the E-loop of CP47 is mediated through its cyclophilin domain. The N-terminal helical domain is closely packed together with the putative C-terminal cyclophilin domain and establishes a strong intramolecular interaction, thereby preventing the access of the cyclophilin domain to other proteins. This was further verified by protein–protein interaction assays using the yeast two-hybrid system. Furthermore, the non-Leucine zipper N-terminal helical bundle contains several new elements for protein–protein interaction that may be of functional significance. Together, this study provides the structure of a plant cyclophilin and explains a possible mechanism for autoinhibition of its function through an intramolecular interaction.     

Transcription regulation of the Saccharomyces cerevisiae PHO5 gene by the Ino2p and Ino4p basic helix-loop-helix proteins

The Saccharomyces cerevisiae PHO5 gene product accounts for a majority of the acid phosphatase activity. Its expression is induced by the basic helix-loop-helix (bHLH) protein, Pho4p, in response to phosphate depletion. Pho4p binds predominantly to two UAS elements (UASp1 at -356 and UASp2 at -247) in the PHO5 promoter. Previous studies from our lab have shown cross-regulation of different biological processes by bHLH proteins. This study tested the ability of all yeast bHLH proteins to regulate PHO5 expression and identified inositol-mediated regulation via the Ino2p/Ino4p bHLH proteins. Ino2p/Ino4p are known regulators of phospholipid biosynthetic genes. Genetic epistasis experiments showed that regulation by inositol required a third UAS site (UASp3 at -194). ChIP assays showed that Ino2p:Ino4p bind the PHO5 promoter and that this binding is dependent on Pho4p binding. These results demonstrate that phospholipid biosynthesis is co-ordinated with phosphate utilization via the bHLH proteins.     

Bio-informatics based analysis of genes implicated in alcohol mediated liver injury

Alcohol induced liver injury has been studied extensively. Using literature search and bioinformatics tools, the present study characterizes the genes involved in alcohol induced liver injury. The cellular and metabolic processes in which genes involved in alcohol induced liver injury are implicated are also discussed. The genes related to alcohol induced liver injury are also involved in affecting certain molecular functions and metabolism of drugs, besides being associated with diseases. In conclusion, the changes in regulation of genes implicated in alcohol induced liver injury apart from causing alcohol mediated hepatic dysfunction may affect other vital processes in the body.     

The Transcription Factor Wilms Tumor 1 Confers Resistance in Myeloid Leukemia Cells against the Proapoptotic Therapeutic Agent TRAIL (Tumor Necrosis Factor α-related Apoptosis-inducing Ligand) by Regulating the Antiapoptotic Protein Bcl-xL

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is considered a promising cancer therapeutic agent due to its ability to induce apoptosis in a variety of cancer cells, while sparing normal cells. However, many human tumors including acute myeloid leukemia (AML) are partially or completely resistant to monotherapy with TRAIL, limiting its therapeutic utility. Therefore, identification of factors that contribute to TRAIL resistance may facilitate future development of more effective TRAIL-based cancer therapies. Here, we report a previously unknown role for WT1 in mediating TRAIL resistance in leukemia. Knockdown of WT1 with shRNA rendered TRAIL-resistant myeloid leukemia cells sensitive to TRAIL-induced cell death, and re-expression of shRNA-resistant WT1 restored TRAIL resistance. Notably, TRAIL-mediated apoptosis in WT1-silenced cells was largely due to down-regulation of the antiapoptotic protein Bcl-xL. Moreover, WT1 expression strongly correlated with overexpression of Bcl-xL in AML cell lines and blasts from AML patients. Furthermore, we found that WT1 transactivates Bcl-xL by directly binding to its promoter. We previously showed that WT1 is a novel client protein of heat shock protein 90 (Hsp90). Consistent with this, pharmacological inhibition of Hsp90 resulted in reduced WT1 and Bcl-xL expression leading to increased sensitivity of leukemia cells to TRAIL-mediated apoptosis. Collectively, our results suggest that WT1-dependent Bcl-xL overexpression contributes to TRAIL resistance in myeloid leukemias.     

Peptide Similarity Search Based and Virtual Screening Based Strategies to Identify Small Molecules to Inhibit CarD–RNAP Interaction in M. tuberculosis

International Journal of Peptide Research and Therapeutics - Inhibition of protein–protein interaction is considered as an innovative approach in the drug development field. In the present...     

Persistent expression of stabilized beta-catenin delays maturation of radial glial cells into intermediate progenitors

Transgenic mice expressing stabilized beta-catenin in neural progenitors develop enlarged brains resulting from increased progenitor expansion. To more precisely define beta-catenin regulation of progenitor fate, we employed a conditional transgenic approach to delete the beta-catenin regulatory domain from neural progenitors, resulting in expression of stabilized protein from its endogenous promoter in these cells and their progeny. An increased fraction of transgenic cortical cells express the progenitor markers Nestin and LewisX, confirming a relative expansion of this population. Sustained beta-catenin activity expands RC2 and Pax6 expression in the developing cortex while postponing the onset of Tbr2 expression, suggesting a delay in maturation of radial glia into intermediate progenitors. Furthermore, transgenic cortical cells fail to either upregulate ErbB4 or develop a mitogenic response to epidermal growth factor, changes that normally accompany the acquisition of an intermediate fate. Likewise, transgenic brains do not develop a distinct subventricular zone or superficial cortical layers, and overexpression of stabilized beta-catenin by in utero electroporation caused a relative reduction of upper layer vs. lower layer cortical neurons, indicating that persistent beta-catenin activity interferes with the generation of progenitors responsible for the production of upper layer cortical neurons. Collectively, these findings demonstrate that beta-catenin functions to maintain the radial glial population, and suggest that downregulation of beta-catenin signaling may be critical to facilitate the transition to an intermediate progenitor phenotype.     

Housekeeping sortase facilitates the cell wall anchoring of pilus polymers in Corynebacterium diphtheriae

Many surface proteins in Gram-positive bacteria are covalently linked to the cell wall through a transpeptidation reaction catalysed by the enzyme sortase. Corynebacterium diphtheriae encodes six sortases, five of which are devoted to the assembly of three distinct types of pilus fibres--SrtA for the SpaA-type pilus, SrtB/SrtC for the SpaD-type pilus, and SrtD/SrtE for the SpaH-type pilus. We demonstrate here the function of SrtF, the so-called housekeeping sortase, in the cell wall anchoring of pili. We show that a multiple deletion mutant strain expressing only SrtA secretes a large portion of SpaA polymers into the culture medium, with concomitant decrease in the cell wall-linked pili. The same phenotype is observed with the mutant that is missing SrtF alone. By contrast, a strain that expresses only SrtF displays surface-linked pilins but no polymers. Therefore, SrtF can catalyse the cell wall anchoring of pilin monomers as well as pili, but it does not polymerize pilins. We show that SrtA and SrtF together generate wild-type levels of the SpaA-type pilus on the bacterial surface. Furthermore, by regulating the expression of SpaA in the cell, we demonstrate that the SrtF function becomes critical when the SpaA level is sufficiently high. Together, these findings provide key evidence for a two-stage model of pilus assembly: pilins are first polymerized by a pilus-specific sortase, and the resulting fibre is then attached to the cell wall by either the cognate sortase or the housekeeping sortase.