Neutral lipids and lipase activity for actinorhodin biosynthesis of Streptomyces coelicolor A3(2)

Neutral lipid accumulation during early growth phase of Streptomyces coelicolor A3(2) was essential for the actinorhodin production during later growth. The activities of lipase and isocitrate dehydrogenase were increasing and decreasing, respectively, suggesting that the degradation products of neutral lipids serve actinorhodin biosynthesis as precursors.     

A novel allele of the P-starvation tolerance gene OsPSTOL1 from African rice (Oryza glaberrima Steud) and its distribution in the genus Oryza

Key message

We have developed allele-specific markers for molecular breeding to transfer the PSTOL1 gene from Kasalath to African mega-varieties, including NERICAs, to improve their tolerance to P-deficient soil.

Abstract

The deficiency of phosphorus (P) in soil is a major problem in Sub-Saharan Africa due to general nutrient depletion and the presence of P-fixing soils. Developing rice cultivars with enhanced P efficiency would, therefore, represent a sustainable strategy to improve the livelihood of resource-poor farmers. Recently the Pup1 locus, a major QTL for tolerance to P deficiency in soil, was successfully narrowed-down to a major gene, the protein kinase OsPSTOL1 (P-starvation tolerance), which was found to be generally absent from modern irrigated rice varieties. Our target is to improve the tolerance of African mega-varieties to P deficiency through marker-assisted introgression of PSTOL1. As a first step, we have determined the Pup1 haplotype and surveyed the presence or absence of PSTOL1 and other genes of the Pup1 locus in African mega-varieties, NERICAs (New Rice for Africa) and their Oryza glaberrima parents. Here, we report the presence of a novel PSTOL1 allele in upland NERICAs that was inherited from the O. glaberrima parent CG14. This allele showed a 35 base-pair substitution when aligned to the Kasalath allele, but maintained a fully conserved kinase domain, and is present in most O. glaberrima accessions evaluated. In-silico and marker analysis indicated that many other genes of the Kasalath Pup1 locus were missing in the O. glaberrima genome, including the dirigent-like gene OsPupK20-2, which was shown to be downstream of PSTOL1. We have developed several allele-specific markers for the use for molecular breeding to transfer the PSTOL1 gene from Kasalath to African mega-varieties, including NERICAs.     

Reaction Kinetics of Substrate Transglycosylation Catalyzed by TreX of Sulfolobus solfataricus and Effects on Glycogen Breakdown

We studied the activity of a debranching enzyme (TreX) from Sulfolobus solfataricus on glycogen-mimic substrates, branched maltotetraosyl-β-cyclodextrin (Glc₄-β-CD), and natural glycogen to better understand substrate transglycosylation and the effect thereof on glycogen debranching in microorganisms. The validation test of Glc₄-β-CD as a glycogen mimic substrate showed that it followed the breakdown process of the well-known yeast and rat liver extract. TreX catalyzed both hydrolysis of α-1,6-glycosidic linkages and transglycosylation at relatively high (>0.5 mM) substrate concentrations. TreX transferred maltotetraosyl moieties from the donor substrate to acceptor molecules, resulting in the formation of two positional isomers of dimaltotetraosyl-α-1,6-β-cyclodextrin [(Glc₄)₂-β-CD]; these were 6¹,6³- and 6¹,6⁴-dimaltotetraosyl-α-1,6-β-CD. Use of a modified Michaelis-Menten equation to study substrate transglycosylation revealed that the k_cat and K_m values for transglycosylation were 1.78 × 10³ s⁻¹ and 3.30 mM, respectively, whereas the values for hydrolysis were 2.57 × 10³ s⁻¹ and 0.206 mM, respectively. Also, enzyme catalytic efficiency (the k_cat/K_m ratio) increased as the degree of polymerization of branch chains rose. In the model reaction system of Escherichia coli, glucose-1-phosphate production from glycogen by the glycogen phosphorylase was elevated ∼1.45-fold in the presence of TreX compared to that produced in the absence of TreX. The results suggest that outward shifting of glycogen branch chains via transglycosylation increases the number of exposed chains susceptible to phosphorylase action. We developed a model of the glycogen breakdown process featuring both hydrolysis and transglycosylation catalyzed by the debranching enzyme.     

TAK1-ECSIT-TRAF6 Complex Plays a Key Role in the TLR4 Signal to Activate NF-κB

ECSIT (evolutionarily conserved signaling intermediate in Toll pathways) is known as a multifunctional regulator in different signals, including Toll-like receptors (TLRs), TGF-β, and BMP. Here, we report a new regulatory role of ECSIT in TLR4-mediated signal. By LPS stimulation, ECSIT formed a high molecular endogenous complex including TAK1 and TRAF6, in which ECSIT interacted with each protein and regulated TAK1 activity, leading to the activation of NF-κB. ECSIT-knockdown THP-1 (ECSIT^KD THP-1) cells exhibited severe impairments in NF-κB activity, cytokine production, and NF-κB-dependent gene expression, whereas those were dramatically restored by reintroduction of wild type (WT) ECSIT gene. Interestingly, ECSIT mutants, which lack a specific interacting domain for either TAK1 or TRAF6, could not restore these activities. Moreover, no significant changes in both NF-κB activity and cytokine production induced by TLR4 could be seen in TAK1^KD or TRAF6^KD THP-1 cells transduced by WT ECSIT, strongly suggesting the essential requirement of TAK1-ECSIT-TRAF6 complex in TLR4 signaling. Taken together, our data demonstrate that the ECSIT complex, including TAK1 and TRAF6, plays a pivotal role in TLR4-mediated signals to activate NF-κB.     

Clusterin stimulates the chemotactic migration of macrophages through a pertussis toxin sensitive G-protein-coupled receptor and Gβγ-dependent pathways

Clusterin induces the expression of various chemotactic cytokines including tumor necrosis factor-α (TNF-α) in macrophages and is involved in the cell migration. According to the results of this study, clusterin induced the directional migration (chemotaxis) of macrophages based on a checkerboard analysis. The chemotactic activity of clusterin was prevented by pretreatment with pertussis toxin (PTX), indicating that the G_αi/o-protein coupled receptor (GPCR) was involved in the chemotactic response of clusterin. Clusterin-stimulated chemotaxis was abrogated in a dose-dependent manner by pretreatment with gallein (a G_βγ inhibitor), indicating the involvement of G_βγ released from the GPCR. In addition, inhibitors of phospholipase C (PLC, U73122) and phosphoinositide 3-kinase (PI3K, LY294002), the key targets of G_βγ binding and activation, suppressed chemotactic migration by clusterin. The phosphorylation of Akt induced by clusterin was blocked by pretreatment with gallein or LY294002 but not with U73122, indicating that G_βγ released from the PTX-sensitive G_i protein complex activated PLC and PI3K/Akt signaling pathways separately. The activation of cellular MAP kinases was essential in that their inhibitors blocked clusterin-induced chemotaxis, and G_βγ was required for the activation of MAP kinases because gallein reduced their phosphorylations induced by clusterin. In addition, the inflammation-induced migration of macrophages was greatly reduced in clusterin-deficient mice based on a thioglycollate-induced peritonitis model system. These results suggest that clusterin stimulates the chemotactic migration of macrophages through a PTX-sensitive GPCR and G_βγ-dependent pathways and describe a novel role of clusterin as a chemoattractant of monocytes/macrophages, suggesting that clusterin may serve as a molecular bridge between inflammation and its remodeling of related tissue by recruiting immune cells.     

S6K1 Negatively Regulates TAK1 Activity in the Toll-Like Receptor Signaling Pathway

Transforming growth factor β (TGF-β)-activated kinase 1 (TAK1) is a key regulator in the signals transduced by proinflammatory cytokines and Toll-like receptors (TLRs). The regulatory mechanism of TAK1 in response to various tissue types and stimuli remains incompletely understood. Here, we show that ribosomal S6 kinase 1 (S6K1) negatively regulates TLR-mediated signals by inhibiting TAK1 activity. S6K1 overexpression causes a marked reduction in NF-κB and AP-1 activity induced by stimulation of TLR2 or TLR4. In contrast, S6K1^−/− and S6K1 knockdown cells display enhanced production of inflammatory cytokines. Moreover, S6K1^−/− mice exhibit decreased survival in response to challenge with lipopolysaccharide (LPS). We found that S6K1 inhibits TAK1 kinase activity by interfering with the interaction between TAK1 and TAB1, which is a key regulator protein for TAK1 catalytic function. Upon stimulation with TLR ligands, S6K1 deficiency causes a marked increase in TAK1 kinase activity that in turn induces a substantial enhancement of NF-κB-dependent gene expression, indicating that S6K1 is negatively involved in the TLR signaling pathway by the inhibition of TAK1 activity. Our findings contribute to understanding the molecular pathogenesis of the impaired immune responses seen in type 2 diabetes, where S6K1 plays a key role both in driving insulin resistance and modulating TLR signaling.     

CIIA negatively regulates neuronal cell death induced by oxygen–glucose deprivation and reoxygenation

Brain ischemia causes neuronal injury leading to stroke and other related brain diseases. However, the precise mechanism of the ischemia-induced neuronal death remains unclear yet. In this study, we showed that CIIA suppressed neuronal cell death induced by oxygen and glucose deprivation followed by reoxygenation (OGD/R), which mimics ischemia and reperfusion in vivo, in neuroblastoma cell lines as well as primary cortical neurons. Furthermore, CIIA inhibited the OGD/R-induced stimulation of apoptosis signal-regulating kinase 1 (ASK1) and its downstream kinases including c-Jun amino-terminal kinase and p38 kinase, concomitantly blocking ASK1 homo-oligomerization and the binding between ASK1 and TRAF2. CIIA also repressed the OGD/R-induced activation of caspase-3 in neuronal cells. Taken together, our results suggest that CIIA attenuates neurotoxicity caused by OGD/R through inhibiting ASK1-dependent signaling events.     

Synechocystis PCC6803 and PCC6906 dnaK2 expression confers salt and oxidative stress tolerance in Arabidopsis via reduction of hydrogen peroxide accumulation

Abiotic stress slows plant growth and development. Because salt stress, particularly from NaCl, acts as an important limiting factor in agricultural productivity, the identification and manipulation of genes related to salt tolerance could improve crop productivity. Prokaryotic, heat shock protein (Hsp), DnaK from the ubiquitous Hsp70 family is upregulated in cells that are under abiotic stress. Synechocystis spp. cyanobacteria encode at least three potential DnaK proteins in their genome. Here, expressions of dnaK1s and dnaK2s from two Synechocystis spp. PCC6803 (Sy6803) and PCC6906 (Sy6906), enhanced salt tolerance in a dnaK-defective Escherichia coli strain. In contrast, dnaK3s in both strains were ineffective, indicating that dnaK3 is functionally different from dnaK1 and dnaK2 in Synechocystis spp. under salt stress. Ectopic expression of dnaK2s from Sy6803 and Sy6906 conferred salt tolerance in transgenic Arabidopsis plants, which exhibited greater root length, chlorophyll content, fresh weight, and survival rate than wild type plants, all in the presence of NaCl. In transgenic plants, hydrogen peroxide (H₂O₂) accumulation was reduced under NaCl stress and loss of chlorophyll content was reduced under H₂O₂ stress. Overall results suggest that dnaK2s from Sy6803 and Sy6906 confer salt and oxidative tolerance in transgenic plants by reduction of H₂O₂ accumulation.