Structural insights into the negative regulation of BRI1 signaling by BRI1-interacting protein BKI1

Brassinosteroids (BRs) are essential steroid hormones that have crucial roles in plant growth and development. BRs are perceived by the cell-surface receptor-like kinase brassinosteroid insensitive 1 (BRI1). In the absence of BRs, the cytosolic kinase domain (KD) of BRI1 is inhibited by its auto-inhibitory carboxyl terminus, as well as by interacting with an inhibitor protein, BRI1 kinase inhibitor 1 (BKI1). How BR binding to the extracellular domain of BRI1 leads to activation of the KD and dissociation of BKI1 into the cytosol remains unclear. Here we report the crystal structure of BRI1 KD in complex with the interacting peptide derived from BKI1. We also provide biochemical evidence that BRI1-associated kinase 1 (BAK1) plays an essential role in initiating BR signaling. Steroid-dependent heterodimerization of BRI1 and BAK1 ectodomains brings their cytoplasmic KDs in the right orientation for competing with BKI1 and transphosphorylation.     

细胞色素P450 1A1和1B1靶向抗肿瘤前药研究进展

细胞色素P450（CYP）能催化各种内源性及外源性化合物的代谢,与多种肿瘤发生有关。其中CYP1A1参与多种前致癌物和致突变物的代谢活化,CYP1B1被认为在许多人癌细胞中特异性表达,参与药物的氧化代谢和前药的活化。CYP1A1和181已成为靶向抗肿瘤前药研究的新靶点。相继有大量相关研究报道,本文就近年来文献报道的CYP1A1和1B1靶向抗肿瘤前药研究进展。     

Roles for N-glycosylation in the dynamics of Edg-1/S1P1 in sphingosine 1-phosphate-stimulated cells

Sphingosine 1-phosphate (Sph-1-P) is a bioactive lipid mediator released from activated platelets. To date, 5 seven-transmembrane-spanning receptors, Edg-1/S1P1, Edg-3/S1P3, Edg-5/S1P2, Edg-6/S1P4 and Edg-8/S1P5, have been identified as specific Sph-1-P receptors. Our recent novel studies established that Edg-1/S1P1 is glycosylated in its N-terminal extracellular portion and further identified the specific glycosylation site as asparagine 30. We also demonstrated that the structure of the N-terminal ectodomain of Edg-1/S1P1 affects both its transport to the cell surface and the N-glycosylation process. These studies revealed a possible regulatory role for the N-glycan on Edg-1/S1P1 in the dynamics of the receptor, such as its lateral and internal movements within the membrane, in ligand-stimulated mammalian cells. Published in 2004.     

CYP1A1基因单核苷酸多态性与肺癌的相关性研究

目的:探讨代谢酶CYP1A1基因MspI位点多态性与新疆汉族人群肺癌遗传易感性之间的相关性.方法:应用聚合酶链式反应(PCR)-限制性片段长度多态性(RFLP)技术检测59例新疆汉族肺癌和84例新疆汉族健康人的CYP1A1基因MspI位点多态性分布频率,并分析了CYP1A1基因MspI位点多态性与新疆汉族人群肺癌遗传易感性和患者性别之间的相关性.结果:(1)CYP1A1基因MspI位点3种多态基因型分布频率在两组间比较差异有统计学意义(χ2=6.682,P=0.035),CC基因型在病例组的分布频率显著高于正常对照组.(2)携带突变CC基因型的个体较携带TT基因型的个体患肺癌的危险性增加(OR=3.759.95%CI=1.228-11.494,P=0.035).(3)男女肺癌患者的CYP1A1基因MspI位点基因型及等位基因频率的差异均无显著性(P>0.05).结论:(1)CC突变基因型可能是新疆汉族人群的肺癌易感因素.(2)CYP1A1基因MspI位点多态性可能与新疆汉族肺癌患者的性别无关.     

Expression of NYV1 encoding the negative regulator of Pmc1 is repressed by two transcriptional repressors,Nrg1 and Mig1

ESCRT components function to form multivesicular bodies for sorting of proteins destined to the yeast vacuole. The calcium hypersensitivity of ESCRT mutants is mainly due to repressed expression of PMR1 through the Rim101/Nrg1 pathway in budding yeast. Here, we show that overexpression of PMC1 and its negative regulator gene NYV1 suppresses and increases calcium hypersensitivity of ESCRT mutants, respectively. Consistently, deletion of NYV1 suppresses their calcium hypersensitivity. Expression of NYV1 is dramatically reduced in ESCRT mutants. Promoter analysis demonstrates that both Nrg1 and Mig1 repress NYV1 expression. Deletion of ESCRTs increases Nrg1 binding, but not Mig1-binding, to the NYV1 promoter. Deletion of MIG1 increases calcium sensitivity of ESCRT mutants due to derepression of NYV1 expression.     

Phosphorylation of CHO1 by Lats1/2 regulates the centrosomal activation of LIMK1 during cytokinesis

Large tumor suppressor 1 and 2 (Lats1/2) regulate centrosomal integrity, chromosome segregation and cytokinesis. As components of the centralspindlin complex, the kinesin-like protein CHO1 and its splicing variant MKLP1 colocalize with chromosome passenger proteins and GTPases and regulate the formation of the contractile ring and cytokinesis; however, the regulatory mechanisms of CHO1/MKLP1 remain elusive. Here, we show that Lats1/2 phosphorylate Ser716 in the F-actin-interacting region of CHO1, which is absent in MKLP1. Phosphorylated CHO1 localized to the centrosomes and midbody, and the actin polymerization factor LIM-kinase 1 (LIMK1) was identified as its binding partner. Overexpression of constitutively phosphorylated and non-phosphorylated CHO1 altered the mitotic localization and activation of LIMK1 at the centrosomes in HeLa cells, leading to the inhibition of cytokinesis through excessive phosphorylation of Cofilin and mislocalization of Ect2. These results suggest that Lats1/2 stringently control cytokinesis by regulating CHO1 phosphorylation and the mitotic activation of LIMK1 on centrosomes.     

Liver kinase B1 regulates the centrosome via PLK1

Liver kinase B1 (LKB1) is a tumor suppressor mutationally inactivated in Peutz–Jeghers syndrome (PJS) and various sporadic cancers. Although LKB1 encodes a kinase that possesses multiple functions, no individual hypothesis posed to date has convincingly explained how loss of LKB1 contributes to carcinogenesis. In this report we demonstrated that LKB1 maintains genomic stability through the regulation of centrosome duplication. We found that LKB1 colocalized with centrosomal proteins and was situated in the mitotic spindle pole. LKB1 deficiency-induced centrosome amplification was independent of AMP-activated protein kinase (AMPK), a well-defined substrate of LKB1. Cells lacking LKB1 exhibited an increase in phosphorylated and total Polo-like kinase 1 (PLK-1), NIMA-related kinase 2 (NEK2), and ninein-like protein (NLP). Overexpression of active PLK1 (T210D) reversed the inhibition of LKB1 on centrosome amplification. In contrast, depletion of PLK1 with siRNA or suppression of PLK1 kinase activity with BTO-1 (5-Cyano-7-nitro-2-benzothiazolecarboxamide-3-oxide) abrogated LKB1 deficiency-induced centrosome amplification. We further characterized that LKB1 phosphorylated and activated AMPK-related kinase 5 (NUAK1 or ARK5) that in turn increased the phosphorylation of MYPT1, enhanced the binding between MYPT1–PP1 and PLK1, and conferred an effective dephosphorylation of PLK1. More importantly, we noted that LKB1-deficient cells exhibited multiple nuclear abnormalities, such as mitotic delay, binuclear, polylobed, grape, large, and micronuclear. Immediate depletion of LKB1 resulted in the accumulation of multiploidy cells. Expression of LKB1 is reversely correlated with the levels of PLK1 in human cancer tissues. Thus, we have uncovered a novel function of LKB1 in the maintenance of genomic stability through the regulation of centrosome mediated by PLK1.     

NP c 1L l 在高脂血症和动脉粥样硬化中的表达分析

目的：研究NPC1L1（Niemann-Pick C1 Like 1）mRNA在单纯高脂血症大鼠和动脉粥样硬化大鼠小肠组织中的表达与差异,探讨其与脂质代谢和动脉粥样硬化之间的关系。方法：通过半定量RT-PCR方法分别检测正常普食组、单纯高脂饲养组和动脉粥样大鼠组小肠组织中NPC1L1 mRNA的表达差异。结果：三个组别大鼠小肠组织中均存在NPC1L2 mRNA,单纯高脂饮食和动脉粥样大鼠小肠组织中NPC1L1 mRNA表达明显高于正常对照大鼠（P〈0．01）;单纯高脂饮食和动脉粥样大鼠小肠组织中NPC1L1 mRNA表达之间无明显差异（P〉0．05）。结论：血脂代谢紊乱与小肠组织中NPC1L1的高表达有关,NPC1L1可能参与了血脂紊乱的病理生理过程;NPC1L1与促成动脉粥样硬化的发生无明显相关性。     

Cex1p facilitates Rna1p-mediated dissociation of the Los1p-tRNA-Gsp1p-GTP export complex

Nuclear tRNA export plays an essential role in key cellular processes such as regulation of protein synthesis, cell cycle progression, response to nutrient availability and DNA damage and development. Like other nuclear export processes, assembly of the nuclear tRNA export complex in the nucleus is dependent on Ran-GTP/Gsp1p-GTP, and dissociation of the export receptor-tRNA-Ran-GTP/Gsp1p-GTP complex in the cytoplasm requires RanBP1/Yrb1p and RanGAP/Rna1p to activate the GTPase activity of Ran-GTP/Gsp1p-GTP. The Saccharomyces cerevisiae Cex1p and Human Scyl1 have also been proposed to participate in unloading of the tRNA export receptors at the cytoplasmic face of the nuclear pore complex (NPC). Here, we provide evidence suggesting that Cex1p is required for activation of the GTPase activity of Gsp1p and dissociation of the receptor-tRNA-Gsp1p export complex in S. cerevisiae. The data suggest that Cex1p recruits Rna1p from the cytoplasm to the NPC and facilitates Rna1p activation of the GTPase activity of Gsp1p by enabling Rna1p to gain access to Gsp1p-GTP bound to the export receptor tRNA complex. It is possible that this tRNA unloading mechanism is conserved in evolutionarily diverse organisms and that other Gsp1p-GTP-dependent export processes use a pathway-specific component to recruit Rna1p to the NPC.     

Fluorescence-based assays for the assessment of drug interaction with the human transporters OATP1B1 and OATP1B3

Hepatic disposition plays a significant role in the pharmacokinetics and pharmacodynamics of a variety of drugs. Sinusoidal membrane transporters have been shown to participate in the hepatic disposition of many pharmaceuticals. Two sinusoidal membrane transporters with an established role in hepatic disposition are OATP1B1 and OATP1B3 (organic anion-transporting polypeptides 1B1 and 1B3, respectively). OATP1B1 and OATP1B3 have been implicated in the hepatic uptake of statin drugs, and polymorphisms linked to OATP1B1 have been associated with deleterious patient endpoints. As a result, OATP1B1 and OATP1B3 represent sites for potential drug-drug interactions. Numerous methods exist for identifying potential drug-drug interactions with transporters. However, relatively few offer the convenience and speed of fluorescence-based assays. Here a fluorescence-based assay was developed for measuring the OATP1B1- and OATP1B3-mediated transport of 8-fluorescein-cAMP (8-FcA). The OATP1B1- and OATP1B3-mediated transport of 8-FcA was time dependent and saturable (K_m = 2.9 and 1.8 μM, V_max = 0.20 and 0.33 pmol/min/cm², respectively). Molecules known to interact with OATPs, including cyclosporin A, rifampicin, and glibenclamide, each demonstrated concentration-dependent inhibition of 8-FcA transport by OATP1B1 and OATP1B3. The in vitro fluorescence-based assays described here using 8-FcA as the substrate are convenient and rapid and have utility in screening drug candidates for potential drug-drug interactions with OATP1B1 and OATP1B3.     

Human Yip1A specifies the localization of Yif1 to the Golgi apparatus

Yip1p and Yif1p are essential for transport from ER to Golgi stack during the early secretory pathway in budding yeast. Here, we report the identification and characterization of human Yif1. Sequence analysis revealed that human Yif1 (HsYif1), like most of the other YIP1 protein family members, contains multiple transmembrane segments. Double immunofluorescence study revealed co-distribution of HsYif1 with Golgi marker such as GS27. To delineate the function of HsYif1, we conducted a yeast two-hybrid assay and identified an interaction between human HsYif1 and HsYip1A, a homolog of yeast Yip1. In addition, our immunoprecipitation pull-down assay validates the interaction between HsYif1 and HsYip1A. Moreover, our immunofluorescence study demonstrates the co-distribution of HsYif1 and HsYip1A. Significantly, over-expression of mutant HsYip1A-lacked cytosolic region disrupts the localization of HsYif1 to the Golgi, suggesting that HsYip1A specifies the localization of HsYif1 to the Golgi. Therefore, we conclude that human Yip1A interacts with and determines the localization of HsYif1 to the Golgi apparatus.