The impact of a single-nucleotide mutation of <Emphasis Type="Italic">bgl2</Emphasis> on cellulase induction in a <Emphasis Type="Italic">Trichoderma reesei</Emphasis> mutant

Background

The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.

Results

To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.

Conclusion

We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

     

PCTAIRE1-Knockdown Sensitizes Cancer Cells to TNF Family Cytokines

While PCTAIRE1/PCTK1/Cdk16 is overexpressed in malignant cells and is crucial in tumorigenesis, its function in apoptosis remains unclear. Here we investigated the role of PCTAIRE1 in apoptosis, especially in the extrinsic cell death pathway. Gene-knockdown of PCTAIRE1 sensitized prostate cancer PPC1 and Du145 cells, and breast cancer MDA-MB-468 cells to TNF-family cytokines, including TNF-related apoptosis-inducing ligand (TRAIL). Meanwhile, PCTAIRE1-knockdown did not sensitize non-malignant cells, including diploid fibroblasts IMR-90 and the immortalized prostate epithelial cell line 267B1. PCTAIRE1-knockdown did not up-regulate death receptor expression on the cell surface or affect caspase-8, FADD and FLIP expression levels. PCTAIRE1-knockdown did promote caspase-8 cleavage and RIPK1 degradation, while RIPK1 mRNA knockdown sensitized PPC1 cells to TNF-family cytokines. Furthermore, the kinase inhibitor SNS-032, which inhibits PCTAIRE1 kinase activity, sensitized PPC1 cells to TRAIL-induced apoptosis. Together these results suggest that PCTAIRE1 contributes to the resistance of cancer cell lines to apoptosis induced by TNF-family cytokines, which implies that PCTAIRE1 inhibitors could have synergistic effects with TNF-family cytokines for cytodestruction of cancer cells.     

Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation

Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.     

Epithelial–mesenchymal transition of A549 cells is enhanced by co-cultured with THP-1 macrophages under hypoxic conditions

Epithelial–mesenchymal transition (EMT) is associated with pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). In this study, we investigated EMT of human pulmonary epithelial-derived cells (A549). A549 cells was either cultured by itself or co-cultured with THP-1 macrophages under normoxic (21% O₂) and hypoxic (2% O₂) conditions. We evaluated the presence of EMT by determining the expression of EMT markers, E-cadherin, vimentin, and fibronectin. To determine the role of TGF-β1 and IL-1β in EMT of the A549 cells, we analyzed the effects of blocking their activity with TGF-β1 inhibitor or IL-1β neutralizing antibody respectively. The A549 cells presented EMT when they were co-cultured with THP-1 macrophages. The EMT of the A549 cells co-cultured with THP-1 macrophages was exacerbated under hypoxia. In addition, the EMT were prevented by the addition of TGF-β1 type I receptor kinase inhibitor. The hypoxic condition increased the mRNA levels of TGF-β1 in A549 cells and THP-1 macrophages and that of IL-1β in THP-1 macrophages when each cells were co-cultured. Anti-IL-1β neutralizing antibody attenuated TGF-β1 secretion in co-culture media under hypoxic conditions. Thus, the IL-1β from THP-1 macrophages up-regulated the TGF-β1 from A549 cells and THP-1 macrophages, and then the TGF-β1 from both cells induced and promoted the EMT of A549 cells when they were co-cultured under hypoxia. Together, these results demonstrate that the interaction between type II pneumocytes and macrophages under hypoxia is necessary for the development of pulmonary fibrosis.     

Active and inactive state structures of unliganded Lactobacillus casei allosteric L‐lactate dehydrogenase

Lactobacillus casei L ‐lactate dehydrogenase (LCLDH) is activated through the homotropic and heterotropic activation effects of pyruvate and fructose 1,6‐bisphosphate (FBP), respectively, and exhibits unusually high pH‐dependence in the allosteric effects of these ligands. The active (R) and inactive (T) state structures of unliganded LCLDH were determined at 2.5 and 2.6 Å resolution, respectively. In the catalytic site, the structural rearrangements are concerned mostly in switching of the orientation of Arg171 through the flexible intersubunit contact at the Q‐axis subunit interface. The distorted orientation of Arg171 in the T state is stabilized by a unique intra‐helix salt bridge between Arg171 and Glu178, which is in striking contrast to the multiple intersubunit salt bridges in Lactobacillus pentosus nonallosteric L ‐lactate dehydrogenase. In the backbone structure, major structural rearrangements of LCLDH are focused in two mobile regions of the catalytic domain. The two regions form an intersubunit linkage through contact at the P‐axis subunit interface involving Arg185, replacement of which with Gln severely decreases the homotropic and hetertropic activation effects on the enzyme. These two regions form another intersubunit linkage in the Q‐axis related dimer through the rigid NAD‐binding domain, and thus constitute a pivotal frame of the intersubunit linkage for the allosteric motion, which is coupled with the concerted structural change of the four subunits in a tetramer, and of the binding sites for pyruvate and FBP. The unique intersubunit salt bridges, which are observed only in the R state structure, are likely involved in the pH‐dependent allosteric equilibrium. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Chimpanzee mothers at Bossou, Guinea carry the mummified remains of their dead infants

The forests surrounding Bossou, Guinea, are home to a small, semi-isolated chimpanzee community studied for over three decades [1]. In 1992, Matsuzawa [2] reported the death of a 2.5-year-old chimpanzee (Jokro) at Bossou from a respiratory illness. The infant's mother (Jire) carried the corpse, mummified in the weeks following death, for at least 27 days. She exhibited extensive care of the body, grooming it regularly, sharing her day- and night-nests with it, and showing distress whenever they became separated. The carrying of infants' corpses has been reported from a number of primate species, both in captivity and the wild [3-7] - albeit usually lasting a few days only - suggesting a phylogenetic continuity for a behavior that is poignant testament to the close mother-infant bond which extends across different primate taxa. In this report we recount two further infant deaths at Bossou, observed over a decade after the original episode but with striking similarities.     

Development of homogeneous luminescence assays for histone demethylase catalysis and binding

Covalent modifications to histones play important roles in chromatin dynamics and the regulation of gene expression. The JumonjiC (JmjC)-containing histone demethylases (HDMs) catalyze the demethylation of methylated lysine residues on histone tails. Here we report the development of homogeneous luminescence-based assay methods for measuring the catalytic activity and the binding affinities of peptides to HDMs. The assays use amplified luminescent proximity homogeneous assay (ALPHA) technology, are sensitive and robust, and can be used for small molecule inhibitor screening of HDMs. We have profiled known inhibitors of JMJD2E and demonstrate a correlation between the inhibitor potencies determined by the ALPHA and other types of assays. Although this study focuses on the JMJD2E isoform, the catalytic turnover and binding assays described here can be used in studies on other HDMs. The assays should be useful for the development of small molecule inhibitors selective for HDM isoforms.     

Bcl-2 and Bcl-XL regulate proinflammatory caspase-1 activation by interaction with NALP1

Caspases are intracellular proteases that cleave substrates involved in apoptosis or inflammation. In C. elegans, a paradigm for caspase regulation exists in which caspase CED-3 is activated by nucleotide-binding protein CED-4, which is suppressed by Bcl-2-family protein CED-9. We have identified a mammalian analog of this caspase-regulatory system in the NLR-family protein NALP1, a nucleotide-dependent activator of cytokine-processing protease caspase-1, which responds to bacterial ligand muramyl-dipeptide (MDP). Antiapoptotic proteins Bcl-2 and Bcl-X(L) bind and suppress NALP1, reducing caspase-1 activation and interleukin-1beta (IL-1beta) production. When exposed to MDP, Bcl-2-deficient macrophages exhibit more caspase-1 processing and IL-1beta production, whereas Bcl-2-overexpressing macrophages demonstrate less caspase-1 processing and IL-1beta production. The findings reveal an interaction of host defense and apoptosis machinery.     

Schnurri-2 controls memory Th1 and Th2 cell numbers in vivo

Schnurri-2 (Shn-2) is a large zinc-finger containing protein, and it plays a critical role in cell growth, signal transduction and lymphocyte development. In Shn-2-deficient CD4 T cells, the activation of NF-kappaB was up-regulated and their ability to differentiate into Th2 cells was enhanced. We herein demonstrate that Th1 and Th2 memory cells are not properly generated from Shn-2-deficient effector Th1/Th2 cells. Even a week after the transfer of effector Th1/Th2 cells into syngeneic mice, a dramatic decrease in the number of Shn-2-deficient donor T cells was detected particularly in the lymphoid organs. The transferred Shn-2-deficient Th1/Th2 cells express higher levels of the activation marker CD69. No significant defect in the BrdU incorporation in the Shn-2-deficient transferred CD4 T cells was observed. The numbers of apoptotic cells were selectively higher in Shn-2-deficient donor Th1/Th2 cell population. Moreover, Shn-2-deficient effector Th1 and Th2 cells showed an increased susceptibility to cell death in in vitro cultures with increased expression of FasL. Transfer of Th2 effector cells over-expressing the p65 subunit of NF-kappaB resulted in a decreased number of p65-expressing cells in the lymphoid organs. As expected, T cell-dependent Ab responses after in vivo immunization of Shn-2-deficient mice were significantly reduced. Thus, Shn-2 appears to control the generation of memory Th1/Th2 cells through a change in their susceptibility to cell death.