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  • 1.

    【2hr】A novel aminopeptidase in the fat body of the moth Achaea janata as a receptor for Bacillus thuringiensis Cry toxins and its comparison with midgut aminopeptidase

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    机译 蛾蛾亚种的脂肪体中一种新型氨肽酶作为苏云金芽孢杆菌Cry毒素的受体及其与中肠氨肽酶的比较
    摘要：Bacillus thuringiensis insecticidal crystal proteins bind to cell-surface receptors which represent a family of aminopeptidases [APN (aminopeptidase N)] present on the brush border membrane of insect midgut cells of susceptible insects leading to pore formation and death of the insect. We report here for the first time the presence of a novel APN in the fat body of the moth Achaea janata. Northern blotting detected at least one APN-specific transcript in the fat body, whereas two transcripts of different sizes were detected in the midgut. We have cloned two full-length APN cDNAs of 3015 bp and 2850 bp from fat body and midgut respectively, which encode proteins of 1004 and 950 amino acids. These two APNs share only 33% amino acid sequence identity, but both display the typical APN features, such as the N-terminal signal peptide, several putative glycosylation sites, C-terminal glycosylphosphatidylinositol anchor signal, the APN-specific zinc-binding/gluzincin motif HEXXHX18E and gluzincin motif GAMENWG. The fat body APN manifested a variation in its expression with respect to tissue and developmental stage. In spite of the abundance of the APN transcript in the fat body, fairly low APN activity was detected in this tissue. The fat-body- and midgut-specific APNs showed differential interaction with various Cry1A toxins. Besides, the level of toxicity of different Cry subtypes varied enormously with mode/site of delivery, such as intrahaemocoelic injections and feeding bioassays. These data indicate that the fat body might be a potential alternative Cry toxin target site in the moth.
  • 2.

    【2hr】Enzymatic cleavage specificity of the proα1(V) chain processing analysed by site-directed mutagenesis

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    机译 通过定点诱变分析proα1（V）链加工的酶切特异性
    摘要：The proteolytic processing of procollagen V is complex and depends on the activity of several enzymes among which the BMP-1 (bone morphogenetic protein-1)/tolloid metalloproteinase and the furin-like proprotein convertases. Few of these processing interactions could have been predicted by analysing the presence of conserved consensus sequences in the proα1(V) chain. In the present study we opted for a cell approach that allows a straightforward identification of processing interactions. A construct encompassing the complete N-terminal end of the proα1(V) chain, referred to as Nα1, was recombinantly expressed to be used for enzymatic assays and for antibody production. Structural analysis showed that Nα1 is a monomer composed of a compact globule and an extended tail, which correspond respectively to the non-collagenous Nα1 subdomains, TSPN-1 (thrombospondin-1 N-terminal domain-like) and the variable region. Nα1 was efficiently cleaved by BMP-1 indicating that the triple helix is not required for enzyme activity. By mutating residues flanking the cleavage site, we showed that the aspartate residue at position P2′ is essential for BMP-1 activity. BMP-1 activity at the C-terminal end of the procollagen V was assessed by generating a furin double mutant (R1584A/R1585A). We showed that, in absence of furin activity, BMP-1 is capable of processing the C-propeptide even though less efficiently than furin. Altogether, our results provide new relevant information on this complex and poorly understood mechanism of enzymatic processing in procollagen V function.
  • 3.

    【2hr】LRRK2 phosphorylates moesin at threonine-558: characterization of how Parkinson's disease mutants affect kinase activity

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    机译 LRRK2在苏氨酸558上磷酸化肌动蛋白：帕金森氏病突变体如何影响激酶活性的表征
    摘要：Mutations in the LRRK2 (leucine-rich repeat kinase-2) gene cause late-onset PD (Parkinson's disease). LRRK2 contains leucine-rich repeats, a GTPase domain, a COR [C-terminal of Roc (Ras of complex)] domain, a kinase and a WD40 (Trp-Asp 40) motif. Little is known about how LRRK2 is regulated, what its physiological substrates are or how mutations affect LRRK2 function. Thus far LRRK2 activity has only been assessed by autophosphorylation and phosphorylation of MBP (myelin basic protein), which is catalysed rather slowly. We undertook a KESTREL (kinase substrate tracking and elucidation) screen in rat brain extracts to identify proteins that were phosphorylated by an activated PD mutant of LRRK2 (G2019S). This led to the discovery that moesin, a protein which anchors the actin cytoskeleton to the plasma membrane, is efficiently phosphorylated by LRRK2, at Thr558, a previously identified in-vivo-phosphorylation site that regulates the ability of moesin to bind actin. LRRK2 also phosphorylated ezrin and radixin, which are related to moesin, at the residue equivalent to Thr558, as well as a peptide (LRRKtide: RLGRDKYKTLRQIRQ) encompassing Thr558. We exploited these findings to determine how nine previously reported PD mutations of LRRK2 affected kinase activity. Only one of the mutations analysed, namely G2019S, stimulated kinase activity. Four mutations inhibited LRRK2 kinase activity (R1941H, I2012T, I2020T and G2385R), whereas the remainder (R1441C, R1441G, Y1699C and T2356I) did not influence activity. Therefore the manner in which LRRK2 mutations induce PD is more complex than previously imagined and is not only caused by an increase in LRRK2 kinase activity. Finally, we show that the minimum catalytically active fragment of LRRK2 requires an intact GTPase, COR and kinase domain, as well as a WD40 motif and a C-terminal tail. The results of the present study suggest that moesin, ezrin and radixin may be LRRK2 substrates, findings that have been exploited to develop the first robust quantitative assay to measure LRRK2 kinase activity.
  • 4.

    【2hr】The role and characterization of phospholipase A1 in mediating lysophosphatidylcholine synthesis in Trypanosoma brucei

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    机译 布鲁氏锥虫中溶血磷脂酰胆碱合成中磷脂酶A1的作用和表征
    摘要：Lysophospholipids are ubiquitous intermediates in a variety of metabolic and signalling pathways in eukaryotic cells. We have reported recently that lysoglycerophosphatidylcholine (lyso-GPCho) synthesis in the insect form of the ancient eukaryote Trypanosoma brucei is mediated by a novel phospholipase A1 (TbPLA1). In the present study, we show that despite equal levels of TbPLA1 gene expression in wild-type insect and bloodstream trypomastigotes, both TbPLA1 enzyme levels and lysoGPCho metabolites are approx. 3-fold higher in the bloodstream form. Both of these parasite stages synthesize identical molecular species of lysoGPCho. TbPLA1 null mutants in the bloodstream form of the parasite are viable, but are deficient in lysoGPCho synthesis, a defect that can be overcome by the expression of an ectopic copy of TbPLA1. The biochemical attributes of TbPLA1-mediated lysoGPCho synthesis were examined in vitro using recombinant TbPLA1. Although TbPLA1 possesses an active-site serine residue, it is insensitive to serine-modifying reagents, such as di-isopropyl fluorophosphate and PMSF, a characteristic shared by lipases that possess lid-sheltered catalytic triads. TbPLA1 does not require metal co-factors for activity, but it does require interfacial activation prior to catalysis. Results from size-exclusion chromatography and binding kinetics analysis revealed that TbPLA1 activation by Triton X-100/GPCho mixed micelle surfaces was not specific and did not require the pre-formation of a specific enzyme–substrate complex to achieve surface binding.
  • 5.

    【2hr】Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors

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    机译 ATP竞争性抑制剂使蛋白激酶C的膜重新分布的机制
    摘要：ATP-competitive inhibitors of PKC (protein kinase C) such as the bisindolylmaleimide GF 109203X, which interact with the ATP-binding site in the PKC molecule, have also been shown to affect several redistribution events of PKC. However, the reason why these inhibitors affect the redistribution is still controversial. In the present study, using immunoblot analysis and GFP (green fluorescent protein)-tagged PKC, we showed that, at commonly used concentrations, these ATP-competitive inhibitors alone induced redistribution of DAG (diacylglycerol)-sensitive PKCα, PKCβII, PKCδ and PKCϵ, but not atypical PKCζ, to the endomembrane or the plasma membrane. Studies with deletion and point mutants showed that the DAG-sensitive C1 domain of PKC was required for membrane redistribution by these inhibitors. Furthermore, membrane redistribution was prevented by the aminosteroid PLC (phospholipase C) inhibitor U-73122, although an ATP-competitive inhibitor had no significant effect on acute DAG generation. Immunoblot analysis showed that an ATP-competitive inhibitor enhanced cell-permeable DAG analogue- or phorbol-ester-induced translocation of endogenous PKC. Furthermore, these inhibitors also enhanced [3H]phorbol 12,13-dibutyrate binding to the cytosolic fractions from PKCα–GFP-overexpressing cells. These results clearly demonstrate that ATP-competitive inhibitors cause redistribution of DAG-sensitive PKCs to membranes containing endogenous DAG by altering the DAG sensitivity of PKC and support the idea that the inhibitors destabilize the closed conformation of PKC and make the C1 domain accessible to DAG. Most importantly, our findings provide novel insights for the interpretation of studies using ATP-competitive inhibitors, and, especially, suggest caution about the interpretation of the relationship between the redistribution and kinase activity of PKC.
  • 6.

    【2hr】Proteasome-mediated CCAAT/enhancer-binding protein δ (C/EBPδ) degradation is ubiquitin-independent

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    机译 蛋白酶体介导的CCAAT /增强子结合蛋白δ（C /EBPδ）降解是泛素依赖性的
    摘要：C/EBPδ (CCAAT/enhancer-binding protein δ) is a member of the C/EBP family of nuclear proteins that function in the control of cell growth, survival, differentiation and apoptosis. We previously demonstrated that C/EBPδ gene transcription is highly induced in G0 growth-arrested mammary epithelial cells but the C/EBPδ protein exhibits a t1/2 of only ∼120 min. The goal of the present study was to investigate the role of C/EBPδ modification by ubiquitin and C/EBPδ proteasome-mediated degradation. Structural and mutational analyses demonstrate that an intact leucine zipper is required for C/EBPδ ubiquitination; however, the leucine zipper does not provide lysine residues for ubiquitin conjugation. C/EBPδ ubiquitination is not required for proteasome-mediated C/EBPδ degradation and the presence of ubiquitin does not increase C/EBPδ degradation by the proteasome. Instead, the leucine zipper stabilizes the C/EBPδ protein by forming homodimers that are poor substrates for proteasome degradation. To investigate the cellular conditions associated with C/EBPδ ubiquitination we treated G0 growth-arrested mammary epithelial cells with DNA-damage- and oxidative-stress-inducing agents and found that C/EBPδ ubiquitination is induced in response to H2O2. However, C/EBPδ protein stability is not influenced by H2O2 treatment. In conclusion, our results demonstrate that proteasome-mediated protein degradation of C/EBPδ is ubiquitin-independent.
  • 7.

    【2hr】Snake-venom-derived Factor IX-binding protein specifically blocks the γ-carboxyglutamic acid-rich-domain-mediated membrane binding of human Factors IX and X

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    机译 蛇毒来源的因子IX结合蛋白可特异性阻断人因子IX和X的富含γ-羧基谷氨酸的结构域介导的膜结合
    摘要：A potent anticoagulant protein, IX-bp (Factor IX binding protein), has been isolated from the venom of Trimeresurus flavoviridis (habu snake) and is known to bind specifically to the Gla (γ-carboxyglutamic acid-rich) domain of Factor IX. To evaluate the molecular basis for its anticoagulation activity, we assessed its interactions with various clotting factors. We found that the anticoagulation activity is primarily due to binding to the Gla domains of Factors IX and X, thus preventing these factors from recognizing phosphatidylserine on the plasma membrane. The present study suggests that ligands that bind to the Gla domains of Factors IX and X may have the potential to become novel anticoagulants.
  • 8.

    【2hr】Transcriptional regulation of the distal promoter of the rat pyruvate carboxylase gene by hepatocyte nuclear factor 3β/Foxa2 and upstream stimulatory factors in insulinoma cells

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    机译 胰岛素细胞中肝细胞核因子3β/ Foxa2和上游刺激因子对大鼠丙酮酸羧化酶基因远端启动子的转录调控。
    摘要：PC (pyruvate carboxylase) plays a crucial role in intermediary metabolism including glucose-induced insulin secretion in pancreatic islets. In the present study, we identified two regions of the 1.2 kb distal promoter, the −803/−795 site and the −408/−403 E-box upstream of the transcription start site, as the important cis-acting elements for transcriptional activation of the luciferase reporter gene. Site-directed mutagenesis of either one of these sites in the context of this 1.2 kb promoter fragment, followed by transient transfections in the insulinoma cell line, INS-1, abolished reporter activity by approx. 50%. However, disruption of either the −803/−795 or the −408/−403 site did not affect reporter gene activity in NIH 3T3 cells, suggesting that this promoter fragment is subjected to cell-specific regulation. The nuclear proteins that bound to these −803/−795 and −408/−403 sites were identified by gel retardation assays as HNF3β (hepatocyte nuclear factor 3β)/Foxa2 (forkhead/winged helix transcription factor box2) and USFs (upstream stimulatory factors), USF1 and USF2, respectively. Chromatin immunoprecipitation assays using antisera against HNF3β/Foxa2, USF1 and USF2 demonstrated that endogenous HNF3β/Foxa2 binds to the −803/−795 Foxa2 site, and USF1 and USF2 bind to the −408/−403 E-box respectively in vivo, consistent with the gel retardation assay results. Although there are weak binding sites located at regions −904 and −572 for PDX1 (pancreatic duodenal homeobox-1), a transcription factor that controls expression of β-cell-specific genes, it did not appear to regulate PC expression in INS-1 cells in the context of the 1.2 kb promoter fragment. The results presented here show that Foxa2 and USFs regulate the distal promoter of the rat PC gene in a cell-specific manner.
  • 9.

    【2hr】Polycomb protein Cbx4 promotes SUMO modification of de novo DNA methyltransferase Dnmt3a

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    机译 聚梳蛋白Cbx4促进从头DNA甲基转移酶Dnmt3a的SUMO修饰
    摘要：The ‘de novo methyltransferase’ Dnmt3a (DNA methyltransferase 3a) has been shown to mediate transcriptional repression. Post-translational modification of Dnmt3a by SUMOylation affects its ability to transcriptionally repress. However, very little is known about how the SUMOylation process is regulated. In the present study, we identified a PcG (Polycomb group) protein, Cbx4 (chromobox 4), as a specific interaction partner of Dnmt3a. Co-expression of Cbx4 and SUMO-1 (small ubiquitin-related modifier-1) along with Dnmt3a in transfected cells results in enhanced modification of Dnmt3a with SUMO-1. Purified Cbx4 also promotes SUMOylation of Dnmt3a in vitro. The modification occurs in the N-terminal regulatory region, including the PWWP (Pro-Trp-Trp-Pro) domain. Our results suggest that Cbx4 functions as a SUMO E3 ligase for Dnmt3a and it might be involved in the functional regulation of DNA methyltransferases by promoting their SUMO modification.
  • 10.

    【2hr】Evidence for oxygen as the master regulator of the responsiveness of soluble guanylate cyclase and cytochrome c oxidase to nitric oxide

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    机译 氧作为可溶性鸟苷酸环化酶和细胞色素C氧化酶对一氧化氮反应性的主要调节因子的证据
    摘要：Haem is used as a versatile receptor for redox active molecules; most notably NO (nitric oxide) and oxygen. Three haem-containing proteins, myoglobin, haemoglobin and cytochrome c oxidase, are now known to bind NO, and in all these cases competition with oxygen plays an important role in the biological outcome. NO also binds to the haem group of sGC (soluble guanylate cyclase) and initiates signal transduction through the formation of cGMP in a process that is oxygen-independent. From biochemical studies, it has been shown that sGC is substantially more sensitive to NO than is cytochrome c oxidase, but a direct comparison in a cellular setting under various oxygen levels has not been reported previously. In this issue of the Biochemical Journal, Cadenas and co-workers reveal how oxygen can act as the master regulator of the relative sensitivity of the cytochrome c oxidase and sGC signalling pathways to NO. These findings have important implications for our understanding of the interplay between NO and oxygen in both physiology and the pathology of diseases associated with hypoxia.
  • 11.

    【2hr】Caspase 3 attenuates XIAP (X-linked inhibitor of apoptosis protein)–mediated inhibition of caspase 9

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    机译 Caspase 3减弱XIAP（X连锁凋亡蛋白抑制剂）介导的Caspase 9抑制
    摘要：During apoptosis, the initiator caspase 9 is activated at the apoptosome after which it activates the executioner caspases 3 and 7 by proteolysis. During this process, caspase 9 is cleaved by caspase 3 at Asp330, and it is often inferred that this proteolytic event represents a feedback amplification loop to accelerate apoptosis. However, there is substantial evidence that proteolysis per se does not activate caspase 9, so an alternative mechanism for amplification must be considered. Cleavage at Asp330 removes a short peptide motif that allows caspase 9 to interact with IAPs (inhibitors of apoptotic proteases), and this event may control the amplification process. We show that, under physiologically relevant conditions, caspase 3, but not caspase 7, can cleave caspase 9, and this does not result in the activation of caspase 9. An IAP antagonist disrupts the inhibitory interaction between XIAP (X-linked IAP) and caspase 9, thereby enhancing activity. We demonstrate that the N-terminal peptide of caspase 9 exposed upon cleavage at Asp330 cannot bind XIAP, whereas the peptide generated by autolytic cleavage of caspase 9 at Asp315 binds XIAP with substantial affinity. Consistent with this, we found that XIAP antagonists were only capable of promoting the activity of caspase 9 when it was cleaved at Asp315, suggesting that only this form is regulated by XIAP. Our results demonstrate that cleavage by caspase 3 does not activate caspase 9, but enhances apoptosis by alleviating XIAP inhibition of the apical caspase.
  • 12.

    【2hr】Dsk1p kinase phosphorylates SR proteins and regulates their cellular localization in fission yeast

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    机译 Dsk1p激酶使SR蛋白磷酸化并调节其在裂殖酵母中的细胞定位
    摘要：Evolutionarily conserved SR proteins (serine/arginine-rich proteins) are important factors for alternative splicing and their activity is modulated by SRPKs (SR protein-specific kinases). We previously identified Dsk1p (dis1-suppressing protein kinase) as the orthologue of human SRPK1 in fission yeast. In addition to its similarity of gene structure to higher eukaryotes, fission yeast Schizosaccharomyces pombe is a unicellular eukaryotic organism in which alternative splicing takes place. In the present study, we have revealed for the first time that SR proteins, Srp1p and Srp2p, are the in vivo substrates of Dsk1p in S. pombe. Moreover, the cellular localization of the SR proteins and Prp2p splicing factor is dependent on dsk1+: Dsk1p is required for the efficient nuclear localization of Srp2p and Prp2p, while it promotes the cytoplasmic distribution of Srp1p, thereby differentially influencing the destinations of these proteins in the cell. The present study offers the first biochemical and genetic evidence for the in vivo targets of the SRPK1 orthologue, Dsk1p, in S. pombe and the significant correlation between Dsk1p-mediated phosphorylation and the cellular localization of the SR proteins, providing information about the physiological functions of Dsk1p. Furthermore, the results demonstrate that the regulatory function of SRPKs in the nuclear targeting of SR proteins is conserved from fission yeast to human, indicating a general mechanism of reversible phosphorylation to control the activities of SR proteins in RNA metabolism through cellular partitioning.
  • 13.

    【2hr】Molecular basis for repression of liver X receptor-mediated gene transcription by receptor-interacting protein 140

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    机译 受体相互作用蛋白140抑制肝X受体介导的基因转录的分子基础
    摘要：Similarities in physiological roles of LXR (liver X receptors) and co-repressor RIP140 (receptor-interacting protein 140) in regulating energy homoeostasis and lipid and glucose metabolism suggest that the effects of LXR could at least partly be mediated by recruitment of the co-repressor RIP140. In the present study, we have elucidated the molecular basis for regulation of LXR transcriptional activity by RIP140. LXR is evenly localized in the nucleus and neither the N-terminal domain nor the LBD (ligand-binding domain) is necessary for nuclear localization. Both LXR subtypes, LXRα and LXRβ, interact with RIP140 and co-localize in diffuse large nuclear domains. Interaction and co-localization are dependent on the LBD of the receptor. The C-terminal domain of RIP140 is sufficient for full repressive effect. None of the C-terminal NR (nuclear receptor)-boxes is required for the co-repressor activity, whereas the NR-box-like motif as well as additional elements in the C-terminal region are required for full repressive function. The C-terminal NR-box-like motif is necessary for interaction with LXRβ, whereas additional elements are needed for strong interaction with LXRα. In conclusion, our results suggest that co-repression of LXR activity by RIP140 involves an atypical binding mode of RIP140 and a repression element in the RIP140 C-terminus.
  • 14.

    【2hr】Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA

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    机译 流感病毒小体介导的带有包封质粒DNA的细胞基因转移
    摘要：Reconstituted influenza virosomes (virus membrane envelopes) have been used previously to deliver pDNA (plasmid DNA) bound to their external surface to a variety of target cells. Although high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible to nucleases and could therefore be prone to rapid degradation under in vivo conditions. In the present study, we show a new method for the production of DNA–virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid DCPC (dicaproylphosphatidylcholine) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density-gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles, which appeared as vesicles with spike proteins inserted in their membranes when analysed by electron microscopy. The DNA–virosomes retained the membrane fusion properties of the native influenza virus. The virosome-associated pDNA was completely protected from degradation by nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the virosomes. DNA–virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA–virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery, offering the advantage of complete DNA protection, which is especially important for future in vivo applications.
  • 15.

    【2hr】Activation of brain calcineurin (Cn) by Cu-Zn superoxide dismutase (SOD1) depends on direct SOD1–Cn protein interactions occurring in vitro and in vivo

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    机译 铜锌超氧化物歧化酶（SOD1）对脑钙调神经磷酸酶（Cn）的激活取决于体内和体外发生的直接SOD1-Cn蛋白相互作用
    摘要：Cn (calcineurin) activity is stabilized by SOD1 (Cu-Zn superoxide dismutase), a phenomenon attributed to protection from superoxide (O2•−). The effects of O2•− on Cn are still controversial. We found that O2•−, generated either in vitro or in vivo did not affect Cn activity. Yet native bovine, recombinant human or rat, and two chimaeras of human SOD1–rat SOD1, all activated Cn, but SOD2 (Mn-superoxide dismutase) did not affect Cn activity. There was also a poor correlation between SOD1 dismutase activity and Cn activation. A chimaera of human N-terminal SOD1 and rat C-terminal SOD1 had little detectable dismutase activity, yet stimulated Cn activity the same as full-length human or rat SOD1. Nevertheless, there was evidence that the active site of SOD1 was involved in Cn activation based on the loss of activation following chelation of Cu from the active site of SOD1. Also, SOD1 engaged in the catalysis of O2•− dismutation was ineffective in activating Cn. SOD1 activation of Cn resulted from a 90-fold decrease in phosphatase Km without a change in Vmax. A possible mechanism for the activation of Cn was identified in our studies as the prevention of Fe and Zn losses from the active site of Cn, suggesting a conformation-dependent SOD1–Cn interaction. In neurons, SOD1 and Cn were co-localized in cytoplasm and membranes, and SOD1 co-immunoprecipitated with Cn from homogenates of brain hippocampus and was present in immunoprecipitates as large multimers. Pre-incubation of pure SOD1 with Cn caused SOD1 multimer formation, an indication of an altered conformational state in SOD1 upon interaction with Cn.
  • 16.

    【2hr】Activation of biliverdin-IXα reductase by inorganic phosphate and related anions

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    机译 无机磷酸酯及相关阴离子对胆绿素-IXα还原酶的活化作用
    摘要：The effect of pH on the initial-rate kinetic behaviour of BVR-A (biliverdin-IXα reductase) exhibits an alkaline optimum with NADPH as cofactor, but a neutral optimum with NADH as cofactor. This has been described as dual cofactor and dual pH dependent behaviour; however, no mechanism has been described to explain this phenomenon. We present evidence that the apparent peak of activity observed at neutral pH with phosphate buffer and NADH as cofactor is an anion-dependent activation, where inorganic phosphate apparently mimics the role played by the 2′-phosphate of NADPH in stabilizing the interaction between NADH and the enzyme. The enzymes from mouse, rat and human all exhibit this behaviour. This behaviour is not seen with BVR-A from Xenopus tropicalis or the ancient cyanobacterial enzyme from Synechocystis PCC 6803, which, in addition to being refractory to activation by inorganic phosphate, are also differentiated by an acid pH optimum with both nicotinamide nucleotides.
  • 17.

    【2hr】Runx3 negatively regulates Osterix expression in dental pulp cells

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    机译 Runx3负调节牙髓细胞中的Osterix表达
    摘要：Osterix, a zinc-finger-containing transcription factor, is required for osteoblast differentiation and bone formation. Osterix is also expressed in dental mesenchymal cells of the tooth germ. However, transcriptional regulation by Osterix in tooth development is not clear. Genetic studies in osteogenesis place Osterix downstream of Runx2 (Runt-related 2). The expression of Osterix in odontoblasts overlaps with Runx3 during terminal differentiation in vivo. Runx3 down-regulates Osterix expression in mouse DPCs (dental pulp cells). Therefore the regulatory role of Runx3 on Osterix expression in tooth development was investigated. Enforced expression of Runx3 down-regulated the activity of the Osterix promoter in the human embryonic kidney 293 cell line. When the Runx3 responsive element on the Osterix promoter, located at −713 to −707 bp (site 3, AGTGGTT) relative to the cap site, was mutated, this down-regulation was abrogated. Furthermore, electrophoretic mobility-shift assay and chromatin immunoprecipitation assays in mouse DPCs demonstrated direct functional binding of Runx3 to the Osterix promoter. These results demonstrate the transcriptional regulation of Osterix expression by Runx3 during differentiation of dental pulp cells into odontoblasts during tooth development.
  • 18.

    【2hr】PKCζ protects against UV-C-induced apoptosis by inhibiting acid sphingomyelinase-dependent ceramide production

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    机译 PKCζ通过抑制酸性鞘磷脂酶依赖性神经酰胺的产生来保护UV-C诱导的细胞凋亡
    摘要：In a recent study, we described that UV-C irradiation resulted in redox-dependent activation and relocalization of A-SMase (acid sphingomyelinase) to the external surface of raft membrane microdomains, hydrolysis of SM (sphingomyelin) associated with the plasma membrane outer leaflet, ceramide generation and apoptosis. In the present study, we have investigated the influence of PKCζ (protein kinase Cζ), an atypical form of PKC on this pathway. This study shows that PKCζ overexpression resulted in the abrogation of UV-C-induced A-SMase translocation and activation into the raft microdomains, lack of ceramide generation and apoptosis inhibition. Moreover, PKCζ overexpression resulted in a decrease in UV-C-induced ROS (reactive oxygen species) production, which correlated with increased gene expression level of various antioxidant enzymes, including TRx (thioredoxin), TR (thioredoxin reductase) 1, TR2 and peroxiredoxin 1/TPx2 (thioredoxin peroxidase 2). Importantly, enforced TPx2 gene expression inhibited UV-C-induced A-SMase translocation. Finally, PKCζ inhibition led to a significant reduction in TPx2 protein expression. Altogether, these results suggest that PKCζ interferes with the UV-activated sphingolipid signalling pathway by regulating the TRx system. These findings may have important consequences for UV-induced carcinogenesis and resistance to phototherapy.
  • 19.

    【2hr】Regulation of the renal-specific Na+–K+–2Cl− co-transporter NKCC2 by AMP-activated protein kinase (AMPK)

    包量

    机译 AMP激活的蛋白激酶（AMPK）对肾脏特异性Na + –K + –2Cl−共转运蛋白NKCC2的调节
    摘要：The renal-specific NKCC2 (Na+–K+–2Cl− co-transporter 2) is regulated by changes in phosphorylation state, however, the phosphorylation sites and kinases responsible have not been fully elucidated. In the present study, we demonstrate that the metabolic sensing kinase AMPK (AMP-activated protein kinase) phosphorylates NKCC2 on Ser126 in vitro. Co-precipitation experiments indicated that there is a physical association between AMPK and the N-terminal cytoplasmic domain of NKCC2. Activation of AMPK in the MMDD1 (mouse macula densa-derived 1) cell line resulted in an increase in Ser126 phosphorylation in situ, suggesting that AMPK may phosphorylate NKCC2 in vivo. The functional significance of Ser126 phosphorylation was examined by mutating the serine residue to an alanine residue resulting in a marked reduction in co-transporter activity when exogenously expressed in Xenopus laevis oocytes under isotonic conditions. Under hypertonic conditions no significant change of activity was observed. Therefore the present study identifies a novel phosphorylation site that maintains NKCC2-mediated transport under isotonic or basal conditions. Moreover, the metabolic-sensing kinase, AMPK, is able to phosphorylate this site, potentially linking the cellular energy state with changes in co-transporter activity.
  • 20.

    【2hr】Substrate specificity and catalytic efficiency of aldo-keto reductases with phospholipid aldehydes

    包量

    机译 磷脂醛与醛酮还原酶的底物特异性和催化效率
    摘要：Phospholipid oxidation generates several bioactive aldehydes that remain esterified to the glycerol backbone (‘core’ aldehydes). These aldehydes induce endothelial cells to produce monocyte chemotactic factors and enhance monocyte–endothelium adhesion. They also serve as ligands of scavenger receptors for the uptake of oxidized lipoproteins or apoptotic cells. The biochemical pathways involved in phospholipid aldehyde metabolism, however, remain largely unknown. In the present study, we have examined the efficacy of the three mammalian AKR (aldo-keto reductase) families in catalysing the reduction of phospholipid aldehydes. The model phospholipid aldehyde POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine] was efficiently reduced by members of the AKR1, but not by the AKR6 or the ARK7 family. In the AKR1 family, POVPC reductase activity was limited to AKR1A and B. No significant activity was observed with AKR1C enzymes. Among the active proteins, human AR (aldose reductase) (AKR1B1) showed the highest catalytic activity. The catalytic efficiency of human small intestinal AR (AKR1B10) was comparable with the murine AKR1B proteins 1B3 and 1B8. Among the murine proteins AKR1A4 and AKR1B7 showed appreciably lower catalytic activity as compared with 1B3 and 1B8. The human AKRs, 1B1 and 1B10, and the murine proteins, 1B3 and 1B8, also reduced C-7 and C-9 sn-2 aldehydes as well as POVPE [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphoethanolamine]. AKR1A4, B1, B7 and B8 catalysed the reduction of aldehydes generated in oxidized C16:0-20:4 phosphatidylcholine with acyl, plasmenyl or alkyl linkage at the sn-1 position or C16:0-20:4 phosphatidylglycerol or phosphatidic acid. AKR1B1 displayed the highest activity with phosphatidic acids; AKR1A4 was more efficient with long-chain aldehydes such as 5-hydroxy-8-oxo-6-octenoyl derivatives, whereas AKR1B8 preferred phosphatidylglycerol. These results suggest that proteins of the AKR1A and B families are efficient phospholipid aldehyde reductases, with non-overlapping substrate specificity, and may be involved in tissue-specific metabolism of endogenous or dietary phospholipid aldehydes.
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