The crystal structure of human isopentenyl diphosphate isomerase at 1.7 A resolution reveals its catalytic mechanism in isoprenoid biosynthesis

The essential Saccharomyces cerevisiae pre-messenger RNA splicing protein 24 (Prp24) has four RNA recognition motifs (RRMs) and facilitates U6 RNA base-pairing with U4 RNA during spliceosome assembly. Prp24 is a component of the free U6 small nuclear ribonucleoprotein particle (snRNP) but not the U4/U6 bi-snRNP, and so is thought to be displaced from U6 by U4/U6 base-pairing. The interaction partners of each of the four RRMs of Prp24 and how these interactions direct U4/U6 pairing are not known. Here we report the crystal structure of the first three RRMs and the solution structure of the first two RRMs of Prp24. Strikingly, RRM 2 forms extensive inter-domain contacts with RRMs 1 and 3. These contacts occupy much of the canonical RNA-binding faces (beta-sheets) of RRMs 1 and 2, but leave the beta-sheet of RRM 3 exposed. Previously identified substitutions in Prp24 that suppress mutations in U4 and U6 spliceosomal RNAs cluster primarily in the beta-sheet of RRM 3, but also in a conserved loop of RRM 2. RNA binding assays and chemical shift mapping indicate that a large basic patch evident on the surface of RRMs 1 and 2 is part of a high affinity U6 RNA binding site. Our results suggest that Prp24 binds free U6 RNA primarily with RRMs 1 and 2, which may remodel the U6 secondary structure. The beta-sheet of RRM 3 then influences U4/U6 pairing through interaction with an unidentified ligand.     

M-LDH serves as a regulatory subunit of the cytosolic substrate-channelling complex in vivo

Nucleoside diphosphate kinase A (NDPK-A) regulates the alpha1 isoform of the AMP-activated protein kinase (AMPK alpha1) selectively, independent of [AMP] and surrounding [ATP], by a process termed substrate channelling. Here, we show, using a range of empirically validated biochemical techniques, that the muscle form (M-LDH or LDH-A) and the heart form (H-LDH or LDH-B) of lactate dehydrogenase are physically associated with the liver cytosolic substrate-channelling complex such that M-LDH associates with NDPK-A, AMPK alpha1 and casein kinase 2 (CK2), whereas H-LDH associates with local NDPK-B. We find that the species of LDH bound to the substrate-channelling complex regulates the in vivo enzymatic activities of both AMPK and CK2, and has a downstream effect on the phospho-status of acetyl CoA carboxylase, a key regulator of cellular fat metabolism known to be a part of the cytosolic substrate-channelling complex in vivo. We hypothesise that the regulatory presence of LDH in the complex couples the substrate-channelling mechanism to both the glycolytic and redox states of the cell, allowing for efficient sensing of cell metabolic status, interfacing with the substrate-channelling complex and regulating the enzymatic activities of AMPK and CK2, two critical protein kinases.     

Aphid-repellent pheromone E-β-farnesene is generated in transgenic Arabidopsis thaliana over-expressing farnesyl diphosphate synthase2

Background and Aims Plant-synthesized sesquiterpenes play a pivotal role in chemotactic interactions with insects. Biosynthesis of functionally diverse sesquiterpenes is dependent on the availability of a pool of the precursor farnesyldiphosphate (FDP). In Arabidopsis thaliana, FPS2, encoding cytosolic farnesyldiphosphate synthase, is implicated in the synthesis of cytosolic FDP, but it is not known whether enhanced levels of FDP have a commensurate effect on sesquiterpene-mediated defence responses. This study examined transgenic arabidopsis plants generated to over-express FPS2 in order to determine if any effects could be observed in the response of aphids, Myzus persicae.Methods Transgenic arabidopsis plants were generated to over-express FPS2 to produce FPS2 in either the cytosol or the chloroplasts. Morphochemical analyses of the transgenic plants were carried out to detremine growth responses of roots and shoots, and for GC-MS profiling of sesquiterpenes. Aphid response to hyrdo-distillate extracts and head-space volatiles from transgenic plants was assessed using a bioassay.Key Results Either over-expression of FPS2 in the cytosol or targetting of its translated product to chlorplasts resulted in stimulatory growth responses of transgenic arabidopsis at early and late developmental stages. GC-MS analysis of hydro-distillate extracts from aerial parts of the plants revealed biosynthesis of several novel sesquiterpenes, including E-β-farnesene, an alarm pheromone of aphids. Both entrapped volatiles and hydro-distillate extracts of the transgenic leaves triggered agitation in aphids, which was related to both time and dose of exposure.Conclusions Over-expression of FPS2 in the cytosol and targeting of its translated product to chloroplasts in arabidopsis led to synthesis of several novel sesquiterpenes, including E-β-farnesene, and induced alarm responses in M. persicae. The results suggest a potential for engineering aphid-resistant strains of arabidopsis.     

Human brain nucleoside diphosphate kinase activity is decreased in Alzheimer's disease and Down syndrome

In brain, nucleoside diphosphate kinase (NDPK) and its coding gene, nm23, have been implicated to modulate neuronal cell proliferation, differentiation, and neurite outgrowth. However, a role of NDPK in neurodegenerative diseases has not been reported yet. Using proteomics techniques, we evaluated the protein levels of NDPK-A in seven brain regions from patients with Alzheimer's disease (AD) and Down syndrome (DS) showing AD-like neuropathology. NDPK-A was significantly decreased in brain regions (frontal, occipital, and parietal cortices) of both disorders. Due to the limitation of brain samples, the activity of NDPK was measured in three brain regions (frontal cortex, temporal cortex, and cerebellum). The specific activity of NDPK was significantly decreased in AD (frontal cortex) and DS (frontal and temporal cortices). Since NDPK-B could also drive the activity of NDPK, protein expression levels of both NDPK-A and NDPK-B were studied in frontal cortex by Western blot analysis. NDPK-A was significantly decreased in AD, which was consistent with the results of proteomics. However, NDPK-A was slightly decreased in DS and protein expression levels of NDPK-B in both DS and AD were moderately decreased, without reaching statistical significance. We propose that oxidative modification of NDPK could lead to the decreased activity of NDPK and, subsequently, influence several neuronal functions in neurodegenerative diseases as multifunctional enzyme through several mechanisms.     

Expression, purification and characterization of recombinant (E)-beta-farnesene synthase from Artemisia annua

A cDNA clone (GenBank Accession No. AY835398) encoding a sesquiterpene synthase, (E)-β-farnesene synthase, has been isolated from Artemisia annua L. It contains a 1746-bp open reading frame coding for 574 amino acids (66.9 kDa) with a calculated pI = 5.03. The deduced amino acid sequence is 30-50% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a single product, β-farnesene, from farnesyl diphosphate. The pH optimum for the recombinant enzyme is around 6.5 and the K_m- and k_cat-values for farnesyl diphosphate, is 2.1 μM and 9.5 × 10⁻³ s⁻¹, respectively resulting in the efficiency 4.5 × 10⁻³ M⁻¹ s⁻¹. The enzyme exhibits substantial activity in the presence of Mg²⁺, Mn²⁺ or Co²⁺ but essentially no activity when Zn²⁺, Ni²⁺ or Cu²⁺ is used as cofactor. The concentration required for maximum activity are estimated to 5 mM, 0.5 mM and <10 μM for Mg²⁺, Co²⁺ or Mn²⁺, respectively. Geranyl diphosphate is not a substrate for the recombinant enzyme.     

Light-dependent subcellular localization of nucleoside diphosphate kinase-1 in Neurospora crassa

Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme localized in cellular organelles and distributed in various organs in prokaryotes and eukaryotes. In Neurospora crassa, NDK-1 is suggested to control catalases in response to heat, oxidative stress and light. In this study, we identified the presence of NDK-1 during most developmental stages in submerged mycelia, aerial hyphae, asexual conidia and perithecia, and the localization of it in soluble, mitochondrial, nuclear and membrane fractions in the mycelial cell. A light-dependent localization of NDK-1 was shown by Western blotting and immunohistochemical analysis using anti-NDK-1 antibody. In the mycelia, NDK-1 was compartmentalized on the plasma membrane in darkness, while it was relocated in the cytoplasm under light. These results suggest that NDK-1 protein was translocated from the plasma membrane to cytoplasm in response to light, and may interact with catalase.     

A novel homodimeric geranyl diphosphate synthase from the orchid Phalaenopsis bellina lacking a DD(X)2-4D motif

Geranyl diphosphate (GDP) is the precursor of monoterpenes, which are the major floral scent compounds in Phalaenopsis bellina . The cDNA of P. bellina GDP synthase ( PbGDPS ) was cloned, and its sequence corresponds to the second Asp-rich motif (SARM), but not to any aspartate-rich (Asp-rich) motif. The recombinant PbGDPS enzyme exhibits dual prenyltransferase activity, producing both GDP and farnesyl diphosphate (FDP), and a yeast two-hybrid assay and gel filtration revealed that PbGDPS was able to form a homodimer. Spatial and temporal expression analyses showed that the expression of PbGDPS was flower specific, and that maximal PbGDPS expression was concomitant with maximal emission of monoterpenes on day 5 post-anthesis. Homology modelling of PbGDPS indicated that the Glu-rich motif might provide a binding site for Mg²⁺ and catalyze the formation of prenyl products in a similar way to SARM. Replacement of the key Glu residues with alanine totally abolished enzyme activity, whereas their mutation to Asp resulted in a mutant with two-thirds of the activity of the wild-type protein. Phylogenetic analysis indicated that plant GDPS proteins formed four clades: members of both GDPS-a and GDPS-b clades contain Asp-rich motifs, and function as homodimers. In contrast, proteins in the GDPS-c and GDPS-d clades do not contain Asp-rich motifs, but although members of the GDPS-c clade function as heterodimers, PbGDPS, which is more closely related to the GDPS-c clade proteins than to GDPS-a and GDPS-b proteins, and is currently the sole member of the GDPS-d clade, functions as a homodimer.     

蛹虫草磷酸甲羟戊酸激酶和焦磷酸甲羟戊酸脱羧酶基因的功能分析

【目的】确定蛹虫草甲羟戊酸途径中的2个关键酶——磷酸甲羟戊酸激酶(CmErg8)和焦磷酸甲羟戊酸脱羧酶(CmErg19)的功能及其对麦角甾醇和虫草素含量的影响。【方法】通过生物信息学分析鉴定蛹虫草中CmErg8和CmErg19,并采用酵母互补确定其功能是否保守;以蛹虫草尿嘧啶营养缺陷型CmΔpyrG为背景菌株,利用农杆菌介导的转化方法对CmErg8和CmErg19进行过表达,观察其对麦角甾醇和虫草素含量的影响。【结果】CmErg8和CmErg19不能互补酵母erg8和erg19突变体的温度敏感表型;CmErg8和CmErg19过表达菌株中麦角甾醇和虫草素含量均有所增加,特别是CmErg19基因过表达可以使虫草素含量提升5倍左右。【结论】本研究揭示了蛹虫草CmErg8和CmErg19的功能,并且发现蛹虫草麦角甾醇合成通路基因可能会影响虫草素含量。     

ROS-Ca is associated with mitochondria permeability transition pore involved in surfactin-induced MCF-7 cells apoptosis

The surfactin can inhibit proliferation and induce apoptosis in cancer cells. Moreover, surfactin can induce cell death in human breast cancer MCF-7 cells through mitochondrial pathway. However, the molecular mechanism involved in this pathway remains to be elucidated. Here, the reactive oxygen species (ROS) and Ca²⁺ on mitochondria permeability transition pore (MPTP) activity, and MCF-7 cell apoptosis which induced by surfactin were investigated. It is found that surfactin evoked mitochondrial ROS generation, and the surfactin-induced cell death was prevented by N-acetylcysteine (NAC, an inhibitor of ROS). An increasing cytoplasmic Ca²⁺ concentration was detected in surfactin-induced MCF-7 apoptosis, which was inhibited by 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM, a chelator of calcium). In addition, the relationship between ROS generation and the increase of cytoplasm Ca²⁺ was determined. The results showed that surfactin initially induced the ROS formation, leading to the MPTP opening accompanied with the collapse of mitochondrial membrane potential (ΔΨ_m). Then the cytoplasmic Ca²⁺ concentration increased in virtue of the changes of mitochondrial permeability, which was prevented by BAPTA-AM. Besides, cytochrome c (cyt c) was released from mitochondria to cytoplasm through the MPTP and activated caspase-9, eventually induced apoptosis. In summary, surfactin has notable anti-tumor effect on MCF-7 cells, however, there was no obvious cytotoxicity on normal cells.     

华北绣线菊二萜生物碱抗血小板聚集活性研究

采用Born氏比浊法观察华北绣线菊小叶变种中分离得到的总碱和 9个hetisine型C2 0 二萜生物碱及其衍生物体外对血小板活化因子 (PAF)、花生四烯酸 (AA)和二磷酸腺苷 (ADP)三种诱导剂引起的血小板聚集活性的影响 ,并初步探讨了构效关系。结果表明 ,华北绣线菊小叶变种中总碱对PAF和ADP诱导的血小板聚集均有明显的抑制作用 ;9个hetisine型C2 0 二萜生物碱中 ,有 8个显著抑制PAF诱导的血小板聚集 ,其活性与分子结构明显相关 ;此外 ,hetisine型生物碱及其衍生物对ADP诱导的血小板聚集亦有一定的抑制作用 ,但总碱及生物碱对AA诱导的聚集影响不明显。提示hetisine型C2 0 二萜生物碱具有抗血小板聚集活性。