Leukemia-Associated Mutations in Nucleophosmin Alter Recognition by CRM1: Molecular Basis of Aberrant Transport

Nucleophosmin (NPM) is a nucleocytoplasmic shuttling protein, normally enriched in nucleoli, that performs several activities related to cell growth. NPM mutations are characteristic of a subtype of acute myeloid leukemia (AML), where mutant NPM seems to play an oncogenic role. AML-associated NPM mutants exhibit altered subcellular traffic, being aberrantly located in the cytoplasm of leukoblasts. Exacerbated export of AML variants of NPM is mediated by the nuclear export receptor CRM1, and due, in part, to a mutationally acquired novel nuclear export signal (NES). To gain insight on the molecular basis of NPM transport in physiological and pathological conditions, we have evaluated the export efficiency of NPM in cells, and present new data indicating that, in normal conditions, wild type NPM is weakly exported by CRM1. On the other hand, we have found that AML-associated NPM mutants efficiently form complexes with CRM1HA (a mutant CRM1 with higher affinity for NESs), and we have quantitatively analyzed CRM1HA interaction with the NES motifs of these mutants, using fluorescence anisotropy and isothermal titration calorimetry. We have observed that the affinity of CRM1HA for these NESs is similar, which may help to explain the transport properties of the mutants. We also describe NPM recognition by the import machinery. Our combined cellular and biophysical studies shed further light on the determinants of NPM traffic, and how it is dramatically altered by AML-related mutations.

三叶木通花发育相关基因的结构、功能和进化研究

花是被子植物最关键的创新（innovation）性状。在被子植物的不同类群中，其形态多种多样，尤其以基部真双子叶植物的花形态最为丰富。大量的系统发育分析表明，在核心真双子叶植物起源之前，几个与花发育相关的MADS-box基因亚家族均发生了大尺度的基因重复事件。因此，在被子植物的不同物种中，花发育相关基因的组成并不相同，并且它们经历了不同的进化历史，这意味着这些基因可能以不同的方式调控花的发育。基部真双子叶植物，作为基部被子植物和核心真双子叶植物之间的过渡类群，对于我们理解被子植物花的进化，揭示核心真双子叶植物花的起源以及基部真双子叶植物花多样性分化的分子机制非常重要。本文以基部真双子叶植物三叶木通为研究材料，着重进行了以下研究工作： 1. 花器官发生过程的观察。三叶木通的花为雌雄同序的单性花。而且，根据成熟花的形态，三叶木通的雌花和雄花都只有一轮花被器官，即三个花瓣状的萼片。扫描电镜的观察结果表明：1）在花器官的发生和发育过程中，在萼片和雄蕊原基之间，确实没有花瓣原基或另一轮萼片原基发生。2）雌花和雄花都是以两性花的方式发生发育的。3）单性花是由于在花发育的最后阶段，雌花中雄蕊或者雄花中心皮的退化而产生的。 2. 花发育相关基因的克隆。应用5’/3’ RACE的方法，我们从三叶木通不同发育阶段的混合花芽中共分离到九个与花发育相关的MADS-box基因： AktFL1、AktFL2、AktAP3_1、AktAP3_2、AktAP3_3、AktPI、AktAG1、AktAG2和AktSEP3。 3. A类MADS-box基因的进化。由于A类基因在进化过程中序列结构的改变，再加上取样的限制，使得A类基因间的进化历史一直不能被很好的理解。因此，本文对A类基因的研究从构建该基因亚家族的系统发育树开始。主要结果如下：1）通过扩大在基部真双子叶植物和被子植物其它重要类群的取样，我们的系统发育树基本上反映了现存被子植物的系统发育关系。2）核心真双子叶植物的A类基因由三个分支组成：euFUL、euAP1和AGL79，它们是通过发生在核心真双子叶植物起源之前的两次几乎同时的基因重复事件产生的。3）在基部真双子叶植物中，山龙眼目、毛茛目和黄杨科的A类基因各形成一支。而且，在这些类群内，发生了多次小尺度的独立的基因重复事件。4）来自单子叶植物的FUL-like基因明显地构成一个单系，并且包括三个分支：OsAMDS14、OsMADS15和OsMADS18。它们是由于两次不连续的基因重复事件产生的。5）不同类型的A类基因产物在C末端拥有不同的保守基元。6）从基因组结构上看，所有的A类基因都拥有八个外显子和七个内含子。7）通过对三叶木通中两个FUL-like型基因（AktFL1和AktFL2）表达式样的观察，我们发现它们在叶原基和发育早期的花原基以及发育着的花器官中都有表达。此外，A类基因表达式样的进化分析结果表明被子植物中该类基因的祖先可能具有广泛的功能，既在营养器官中表达又在生殖器官中表达 。 4. B类基因表达式样的保守性和多样性。通过对B类基因的系统发育和表达式样分析，得到以下结果：1）三叶木通中的三个paleoAP3基因是通过两次基因重复事件产生的。2）在木通科或木通属内，PI型基因并没有发生基因重复事件。3）RT-PCR结果表明，AktAP3_1在雌花中的表达量比雄花中高，而AktAP3_2则在雄花中的表达量比雌花中高。AktAP3_3和AktPI在雌花和雄花中的表达水平相似。4）原位杂交分析显示这些基因在发育着的雄蕊和心皮中表达。此外，AktAP3_3和AktPI还在萼片中表达，可能参与花瓣状萼片的发育。 5. 三叶木通C/D和E类基因的序列结构和表达分析。通过序列结构分析，我们发现，与其它被子植物AG同源基因编码的MADS-domain蛋白一样，AktAG1和AktAG2在MADS结构域的N末端都拥有一段氨基酸序列的延伸，AktAG1为20个氨基酸;AktAG2为7个氨基酸。原位杂交分析表明AktAG1和AktAG2主要在发育着的雄蕊和心皮中表达，说明它们具有决定生殖器官发育这一保守的功能。 AktSEP3属于AGL9型的E类基因。该基因在所有花器官中都有表达，说明和其它被子植物的E类基因一样，AktSEP3在三叶木通中对于所有花器官的发育都是必需的。 6. 各类MADS-domain蛋白间的相互作用。在前面工作的基础上，我们首次对三叶木通中上述MADS-domain蛋白间的作用方式进行了研究。酵母双杂交结果表明：1）AktSEP3的C末端具有转录激活功能。2）三个AktAP3蛋白与AktPI蛋白都能够形成异源二聚体，但是它们之间的作用能力并不相同。3）AktSEP3蛋白可以与AktFL1、AktPI、AktAG1和AktAG2形成异源二聚体，充分体现了E类基因产物作用式样的保守性。4）AktFL1与AktPI、AktSEP3和AktAG2也能形成异源二聚体，这与核心真双子叶植物的euFUL型蛋白在作用式样上是非常相似的。 综合以上结果，我们探讨了三叶木通花发育的分子机制。在三叶木通的三轮花器官中，与拟南芥等模式植物相似的是：E类（AktSEP3）基因在每一轮花器官中都起作用;此外，A类（AktFL1）和B类（AktAP3_3和AktPI）基因在花瓣状的萼片中有不同程度的表达，类似于拟南芥的第二轮;B类（AktAP3_1、AktAP3_2、AktAP3_3和AktPI）和C/D类（AktAG1和AktAG2）基因在雄蕊的发育过程中起作用;C/D类（AktAG1和AktAG2）基因对心皮的发育起作用。与拟 南芥等模式植物不同的是：1）虽然原位杂交分析表明，AktFL1、AktAP3_3、AktPI和AktSEP3都在花瓣状的萼片中有 不同程度的表达，但是它们的蛋白质产物AktFL1与AktSEP3和AktAP3_3与AktPI都只能形成较弱的异源二聚体。而 且，根据我们的研究结果，在三叶木通中没有找到euAP1型的A类基因，只有两个FUL-like型的A类基因。它们的功能 与核心真双子叶植物中的euFUL型基因相似。因此，AktFL1很可能与其它调控因子共同作用负责花分生组织的形成;AktFL1/AktAG2则可能在花发育的后期起作用。那么，三叶木通花瓣状萼片的发育是否需要AktFL1/AktSEP3和 AktAP3_3/AktPI的参与，还是另有其它转录因子的参与，仍然需要更深入的研究。2）虽然在三叶木通中，雄蕊的发 育同样需要B、C/D和E类基因的参与，但是由于小尺度的基因重复事件，在该物种中只拥有三个paleoAP3型基因，而没有euAP3型基因。而且，由于复制拷贝间的亚功能化，AktAP3_1/AktPI主要参与雌花的发育过程;而AktAP3_2/AktPI主要参与雄花的发育过程。

金粟兰花发育相关基因的结构、功能和进化研究

花是被子植物区别于其它植物大类群的最重要的特征，其形态多种多样。花的发育取决于一个复杂的涉及到多个基因和过程的调控体系，因此花的起源和多样化过程实际上可以理解为这个调控体系的进化过程。在被子植物的不同物种中，花发育相关基因的组成并不相同，且经历了不同的进化历史，这意味着这些基因可能以不同的方式调控花的发育。相对于核心真双子叶植物相对稳定的花形态结构而言，基部被子植物的花具有丰富的多样性。因此，对基部被子植物花发育相关基因的研究对于我们理解被子植物花的进化非常重要。 金粟兰科（Chloranthaceae）是基部被子植物的代表类群之一。与研究得比较深入的模式植物相比，花被、雄蕊或雌蕊的缺失，使得该科植物的花比较简单。因此对该科植物中花发育基因的研究不仅有助于揭示花的起源以及花多样性分化的分子机制，还将为认识花部构造简单化的机制提供资料。本文以金粟兰（Chloranthus spicatus）为实验材料，取得了以下研究结果： 1.花发育相关基因的克隆 应用5’/3’RACE的方法，我们从金粟兰不同发育阶段的混合花芽中克隆到了与花发育相关的MADS-box基因：CsPI、CsAG1和CsAG2。 2. 两个A类MADS-box基因表达式样的对比分析 在营养分生组织向生殖分生组织的转变中，花原基的形成，以及随后雄蕊、心皮、花粉、胚珠和胚囊的发育中，CsAP1-1和 CsAP1-2基因均表达。唯一不同之处在于，在花发育成熟期，CsAP1-1在外珠被也有表达，而CsAP1-2在外珠被处没有表达，而只在内珠被处表达。这一结果反映了基因重复事件发生后，两个基因在功能上也有了一些分化。 3. B类基因功能的保守性和多样性 通过转基因实验和蛋白质相互作用研究对B类基因的功能和作用方式进行了研究，得到以下结果：1）金粟兰CsAP3基因的C末端的点突变所造成的paleoAP3基元的部分缺失对该基因的功能没有决定性的影响;2）金粟兰paleoAP3型基因CsAP3所编码蛋白的C末端以及paleoAP3基元，与拟南芥euAP3型基因AtAP3所编码蛋白的C末端以及euAP3基元没有功能上的不同;3）金粟兰paleoAP3型基因CsAP3与拟南芥euAP3型基因AtAP3 的主要功能存在一定差异，前者主要参与雄蕊形成，而后者既参与雄蕊的形成也参与花瓣的形成; 4）CsPI基因所编码的蛋白可以与AP3类蛋白相互作用进而影响花瓣的形成，因此该基因在功能上是保守的。 4. 金粟兰CsAG1基因的序列结构和功能分析 通过序列结构分析发现，CsAG1属于C类基因，具有保守的AG I基元和AG II基元。过量表达实验分析表明CsAG1的功能与A类基因的功能是相拮抗的。 5. 各类MADS-domain蛋白间的相互作用 在前面工作的基础上，我们首次对金粟兰中各类MADS-domain蛋白间的作用方式进行了研究。酵母双杂交结果表明：1）C末端的完整性对于MADS-domain蛋白二聚体的形成没有影响; 2）去掉M区的CsAP3蛋白与CsPI蛋白都能够形成异源二聚体，同时它们又可以各自形成同源二聚体; 3）E类蛋白既可以和A类或C类基因产物相互作用，也可以同AP3和PI型蛋白相互作用，充分体现了E类基因产物作用式样的保守性; 4）金粟兰中，FUL-like型基因所编码的蛋白CsAP1-1与CsSEP3和CsAG1也能形成异源二聚体，这与核心真双子叶植物的euFUL型蛋白在作用式样上是非常相似的。然而，金粟兰CsAP1-1蛋白不能形成同源二聚体。 综合以上结果发现，在无花被的金粟兰中，仍然存在着A、B、C/D、E类花发育相关的基因。这些基因的功能与核心真双子叶植物中同类基因的相比，有些是保守的，比如CsPI基因可以参与花被的形成;但也有一些是不同的，比如CsAP3基因主要参与雄蕊形成而非花被形成过程。由此可以看出被子植物花器官的发育是一个非常复杂的调控过程，不同植物中的调控机理及进化历程可能是不同的。

Bayesian correlated clustering to integrate multiple datasets.

MOTIVATION: The integration of multiple datasets remains a key challenge in systems biology and genomic medicine. Modern high-throughput technologies generate a broad array of different data types, providing distinct-but often complementary-information. We present a Bayesian method for the unsupervised integrative modelling of multiple datasets, which we refer to as MDI (Multiple Dataset Integration). MDI can integrate information from a wide range of different datasets and data types simultaneously (including the ability to model time series data explicitly using Gaussian processes). Each dataset is modelled using a Dirichlet-multinomial allocation (DMA) mixture model, with dependencies between these models captured through parameters that describe the agreement among the datasets. RESULTS: Using a set of six artificially constructed time series datasets, we show that MDI is able to integrate a significant number of datasets simultaneously, and that it successfully captures the underlying structural similarity between the datasets. We also analyse a variety of real Saccharomyces cerevisiae datasets. In the two-dataset case, we show that MDI's performance is comparable with the present state-of-the-art. We then move beyond the capabilities of current approaches and integrate gene expression, chromatin immunoprecipitation-chip and protein-protein interaction data, to identify a set of protein complexes for which genes are co-regulated during the cell cycle. Comparisons to other unsupervised data integration techniques-as well as to non-integrative approaches-demonstrate that MDI is competitive, while also providing information that would be difficult or impossible to extract using other methods.

人载脂蛋白APOA5的克隆表达纯化及相互作用蛋白研究

人类的载脂蛋白A5（apolipoprotein A5，APOA5）是一个新近发现的载脂蛋白家族成员。它在血浆中的含量比其他载脂蛋白低1-2个数量级，但能显著影响血浆三酰甘油水平，对血脂代谢具有重要意义，可以作为降血脂药物治疗中一个强有力的潜在靶标。 由于APOA5在血浆中含量低，直接从血浆中分离纯化很困难，国内一直没有报道简易可靠的纯化方法。为进一步研究APOA5的生物学特性，探讨其与TG代谢中的其它关键成分之间的相互关系，揭示其在脂类代谢相关疾病中的重要地位，必须有大量的蛋白和抗体用于基础研究。因此本研究首先利用基因工程技术，诱导表达纯化APOA5蛋白，免疫动物制备多克隆抗体，为进一步研究人肝脏细胞中APOA5的相互作用蛋白，研究APOA5蛋白在肝脏细胞中的功能奠定基础。 为了深入研究APOA5在肝脏中如何行使功能，我们采用细菌双杂交技术寻找与APOA5相互作用的蛋白因子。并采用Pull-down技术，免疫荧光及免疫共沉淀技术进一步确证其在体外和体内的相互作用关系，为进一步阐明APOA5在体内的生理功能提供了新的线索。 第一部分 APOA5基因的克隆、原核表达、纯化及其多克隆抗体的制备 本研究首先应用基因克隆技术，从人肝癌细胞系SMMC-7721的cDNA中扩增出1.1 kb的ApoA5基因全长序列。然后将其克隆至表达载体pThioHisD，构建原核表达载体pTH-APOA5。该重组质粒转化至大肠杆菌 BL21（DE3），成功实现人APOA5融合蛋白在大肠杆菌中的表达。经发酵得到高效表达的融合蛋白。 融合蛋白在 IPGT 诱导下以包涵体的形式大量表达。利用融合蛋白上的一段组氨酸序列，用镍离子亲和柱进行纯化和复性后，获得较高纯度的人APOA5融合蛋白。利用该融合蛋白免疫新西兰大耳白兔，获得了高效价的兔抗人APOA5多克隆抗体，Western Blot结果显示此多克隆抗体与APOA5特异性结合。 第二部分 细菌双杂交筛选与APOA5相互作用的蛋白 本实验首先构建了pBT-APOA5重组质粒，经双酶切、PCR和测序鉴定证明重组诱饵质粒构建成功，并进行了表达、自激活鉴定。Western Blot鉴定证实报告菌株中表达了分子量为 68 kD左右的重组融合蛋白，与预测的分子量APOA5（41 kD）/lamda cI （27 kD）一致。自激活实验证明诱饵蛋白不能单独激活报告基因，可用于筛选人肝脏cDNA文库。经过双重抗性筛选和回复筛选，分离出10个阳性克隆。对结果进行生物信息学分析，得到7个与APOA5相互作用的蛋白，其中BI1为细胞凋亡调节因子；ATP6、CYTB、ND2、COX-1为线粒体表达蛋白； ALB、TTR为血清蛋白。 第三部分 APOA5与BI1相互作用的确证 首先构建了BI1的原核表达载体pGEX-5X-3-BI1，利用Pull-down实验检测了APOA5与BI1在体外具有相互作用。然后构建了BI1的真核表达载体pCDNA3.1-HA-BI1和APOA5的真核表达载体pCDNA3.1-APOA5，并验证其表达。通过免疫荧光细胞内共定位研究发现，靶蛋白APOA5主要分布于胞浆，与BI1在HEK293细胞有共定位，即APOA5与BI1存在相互作用的可能。最后利用免疫共沉淀手段，在HEK293细胞中确证了靶蛋白APOA5与BI1在体内的相互作用。 上述研究结果，为深入研究APOA5在体内的生物学功能提供了新的思路。 Apolipoprotein A5 (APOA5) is a newly discovered protein belongs to apolipoprotein family. APOA5’s concentration is 1-2 orders of magnitude lower than other apolipoproteins in the circulation. APOA5 significantly affected plasma triglyceride levels, which is important on lipid metabolism. APOA5 has strong potential to be used as a hypolipidemic drug target. Large amount of APOA5 protein and antibodies are needed in basic research, such as biological characteristics study of the APOA5, its relationship with other key components in TG metabolism, its role played in Lipid metabolism-related diseases. Due to its low concentration in plasma, separation and purification of APOA5 from the plasma is very difficult. Until now no report on simple and reliable method for purification has been published in China. In this study, we firstly got APOA5 recombinant protein using genetic engineering technology. The purified recombinant protein was used to immunize rabbits to get antiserum. It is important for further study of the APOA5 protein-interacting protein. And it lays the foundation for studing APOA5 function in liver. In order to study APOA5 function in liver, we used bacterial two-hybrid technology to find the APOA5 protein interactor. Pull-down, immunofluorescence and immunoprecipitation techniques were used to further confirm the interaction between APOA5 with its interactor in vitro and in vivo. All of these stdudies provided new clues on its physiological functions in vivo. Part I: Cloning, prokaryotic expression, purification and polyclonal antibody preparation of APOA5 First of all, we amplified APOA5 CDS sequence from the human hepatoma cell line SMMC-7721, and subcloned into Expression vector pThioHisD, and got the recombinants named pTH-APOA5. The plasmid was transformed to BL21 (DE3). E. coli BL21(DE3) cells bearing the pTH-APOA5 plasmid were cultured and APOA5 protein synthesis was induced by the addition of IPTG. Recombinant protein was expression in the form of inclusion. Inclusion bodies were dissolved in phosphate-buffered saline containing 8 M urea and 40 mM imidazole, then applied to a Ni2+ affinity column, and were eluted in a buffer containing 4 M urea and 200 mM imidazole. Fractions containing the APOA5 protein were pooled and dialyzed against buffer containing phosphate-buffered saline. Antiserum to recombinant human APOA5 was generated by immuning rabbit. Western Blot showed that this antiserum specific binding with APOA5. Part II Two-hybrid system screening protein interactions with the APOA5 The coding sequence of human APOA5 was amplified using synthetic oligonucleotide primers from pTH-APOA5 vector and was subcloned into the pBT plasmidc to yield pBT-APOA5 vector. DNA sequencing was performed to verify that no unwanted mutations occurred during the process of plasmid vector construction. We verified recombinant protein expression and tested self-activation by pBT-APOA5 prior to screening. Western Blot verified inducing a 68 kD band, consistent with the predicted molecular weight (APOA5 41 kD, lamda cI 27 kD). pBT-APOA5 can be used for screening human liver cDNA library because it can not self-activation. Totally 10 positive clones were isolated. The nucleotide sequence of the positive clones were determined and compared to NCBI nucleotide sequence databases. We got 7 protein which interact with APOA5, included BI1(Apoptosis regulator); ATP6, CYTB, ND2, COX-1(Mitochondrial protein) and ALB, TTR(Serum protein). Part III Confirming of interaction between APOA5 with BI1 pGEX-5X-3-BI1 vector was subcloned at first. Pull-down experiments were used to detect the interaction between APOA5 with BI1 in vitro. Later, pCDNA3.1-HA-BI1 and pCDNA3.1-APOA5 were subcloned. Through immunofluorescence co-localization study, we found APOA5 mainly distributed in the cytoplasm. APOA5 is co-localization with BI1 in HEK293 cells. Finally, we verified interaction between APOA5 with BI1 in vivo through immunoprecipitation.

A transcriptase reversa como alvo terapêutico em doenças retrovirais

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Distinct functions of POT1 at telomeres.

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres.

The F4 fimbrial chaperone FaeE is stable as a monomer that does not require self-capping of its pilin-interactive surfaces.

Many Gram-negative bacteria use the chaperone-usher pathway to express adhesive surface structures, such as fimbriae, in order to mediate attachment to host cells. Periplasmic chaperones are required to shuttle fimbrial subunits or pilins through the periplasmic space in an assembly-competent form. The chaperones cap the hydrophobic surface of the pilins through a donor-strand complementation mechanism. FaeE is the periplasmic chaperone required for the assembly of the F4 fimbriae of enterotoxigenic Escherichia coli. The FaeE crystal structure shows a dimer formed by interaction between the pilin-binding interfaces of the two monomers. Dimerization and tetramerization have been observed previously in crystal structures of fimbrial chaperones and have been suggested to serve as a self-capping mechanism that protects the pilin-interactive surfaces in solution in the absence of the pilins. However, thermodynamic and biochemical data show that FaeE occurs as a stable monomer in solution. Other lines of evidence indicate that self-capping of the pilin-interactive interfaces is not a mechanism that is conservedly applied by all periplasmic chaperones, but is rather a case-specific solution to cap aggregation-prone surfaces.

Homodimerization Is Essential for the Receptor for Advanced Glycation End Products (RAGE)-mediated Signal Transduction

The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that binds to diverse ligands and initiates a downstream proinflammatory signaling cascade. RAGE activation has been linked to diabetic complications, Alzheimer disease, infections, and cancers. RAGE is known to mediate cell signaling and downstream proinflammatory gene transcription activation, although the precise mechanism surrounding receptor-ligand interactions is still being elucidated. Recent fluorescence resonance energy transfer evidence indicates that RAGE may form oligomers on the cell surface and that this could be related to signal transduction. To investigate whether RAGE forms oligomers, protein-protein interaction assays were carried out. Here, we demonstrate the interaction between RAGE molecules via their N-terminal V domain, which is an important region involved in ligand recognition. By protein cross-linking using water-soluble and membrane-impermeable cross-linker bis(sulfosuccinimidyl) suberate and nondenaturing gels, we show that RAGE forms homodimers at the plasma membrane, a process potentiated by S100B and advanced glycation end products. Soluble RAGE, the RAGE inhibitor, is also capable of binding to RAGE, similar to V peptide, as shown by surface plasmon resonance. Incubation of cells with soluble RAGE or RAGE V domain peptide inhibits RAGE dimerization, subsequent phosphorylation of intracellular MAPK proteins, and activation of NF-kappa B pathways. Thus, the data indicate that dimerization of RAGE represents an important component of RAGE-mediated cell signaling.

Bagging statistical network inference from large-scale gene expression data.

Modern biology and medicine aim at hunting molecular and cellular causes of biological functions and diseases. Gene regulatory networks (GRN) inferred from gene expression data are considered an important aid for this research by providing a map of molecular interactions. Hence, GRNs have the potential enabling and enhancing basic as well as applied research in the life sciences. In this paper, we introduce a new method called BC3NET for inferring causal gene regulatory networks from large-scale gene expression data. BC3NET is an ensemble method that is based on bagging the C3NET algorithm, which means it corresponds to a Bayesian approach with noninformative priors. In this study we demonstrate for a variety of simulated and biological gene expression data from S. cerevisiae that BC3NET is an important enhancement over other inference methods that is capable of capturing biochemical interactions from transcription regulation and protein-protein interaction sensibly. An implementation of BC3NET is freely available as an R package from the CRAN repository. © 2012 de Matos Simoes, Emmert-Streib.

Association Study of 167 Candidate Genes for Schizophrenia Selected by a Multi-Domain Evidence-Based Prioritization Algorithm and Neurodevelopmental Hypothesis

Integrating evidence from multiple domains is useful in prioritizing disease candidate genes for subsequent testing. We ranked all known human genes (n = 3819) under linkage peaks in the Irish Study of High-Density Schizophrenia Families using three different evidence domains: 1) a meta-analysis of microarray gene expression results using the Stanley Brain collection, 2) a schizophrenia protein-protein interaction network, and 3) a systematic literature search. Each gene was assigned a domain-specific p-value and ranked after evaluating the evidence within each domain. For comparison to this
ranking process, a large-scale candidate gene hypothesis was also tested by including genes with Gene Ontology terms related to neurodevelopment. Subsequently, genotypes of 3725 SNPs in 167 genes from a custom Illumina iSelect array were used to evaluate the top ranked vs. hypothesis selected genes. Seventy-three genes were both highly ranked and involved in neurodevelopment (category 1) while 42 and 52 genes were exclusive to neurodevelopment (category 2) or highly ranked (category 3), respectively. The most significant associations were observed in genes PRKG1, PRKCE, and CNTN4 but no individual SNPs were significant after correction for multiple testing. Comparison of the approaches showed an excess of significant tests using the hypothesis-driven neurodevelopment category. Random selection of similar sized genes from two independent genome-wide association studies (GWAS) of schizophrenia showed the excess was unlikely by chance. In a further meta-analysis of three GWAS datasets, four candidate SNPs reached nominal significance. Although gene ranking using integrated sources of prior information did not enrich for significant results in the current experiment, gene selection using an a priori hypothesis (neurodevelopment) was superior to random selection. As such, further development of gene ranking strategies using more carefully selected sources of information is warranted.

Interfacing cellular networks of S. cerevisiae and E. coli: Connecting dynamic and genetic information

Background: In recent years, various types of cellular networks have penetrated biology and are nowadays used omnipresently for studying eukaryote and prokaryote organisms. Still, the relation and the biological overlap among phenomenological and inferential gene networks, e.g., between the protein interaction network and the gene regulatory network inferred from large-scale transcriptomic data, is largely unexplored.

Results: We provide in this study an in-depth analysis of the structural, functional and chromosomal relationship between a protein-protein network, a transcriptional regulatory network and an inferred gene regulatory network, for S. cerevisiae and E. coli. Further, we study global and local aspects of these networks and their biological information overlap by comparing, e.g., the functional co-occurrence of Gene Ontology terms by exploiting the available interaction structure among the genes.

Conclusions: Although the individual networks represent different levels of cellular interactions with global structural and functional dissimilarities, we observe crucial functions of their network interfaces for the assembly of protein complexes, proteolysis, transcription, translation, metabolic and regulatory interactions. Overall, our results shed light on the integrability of these networks and their interfacing biological processes.

Using SILAC proteomics to investigate the effect of the mycotoxin, alternariol, in the human H295R steroidogenesis model

The mycotoxin alternariol (AOH) is an important contaminant of fruits and cereal products. The current study sought to address the effect of a non-toxic AOH concentration on the proteome of the steroidogenic H295R cell model. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture (SILAC) coupled to 1D-SDS-PAGE-LC-MS/MS was applied to subcellular-enriched protein samples. Gene ontology (GO) and ingenuity pathway analysis (IPA) were further carried out for functional annotation and identification of protein interaction networks. Furthermore, the effect of AOH on apoptosis and cell cycle distribution was also determined by the use of flow cytometry analysis. This work identified 22 proteins that were regulated significantly. The regulated proteins are those involved in early stages of steroid biosynthesis (SOAT1, NPC1, and ACBD5) and C21-steroid hormone metabolism (CYP21A2 and HSD3B1). In addition, several proteins known to play a role in cellular assembly, organization, protein synthesis, and cell cycle were regulated. These findings provide a new framework for studying the mechanisms by which AOH modulates steroidogenesis in H295R cell model. 

Nuclear translocation and functions of growth factor receptors

Cellular signal transduction in response to environmental signals involves a relay of precisely regulated signal amplifying and damping events. A prototypical signaling relay involves ligands binding to cell surface receptors and triggering the activation of downstream enzymes to ultimately affect the subcellular distribution and activity of DNA-binding proteins that regulate gene expression. These so-called signal transduction cascades have dominated our view of signaling for decades. More recently evidence has accumulated that components of these cascades can be multifunctional, in effect playing a conventional role for example as a cell surface receptor for a ligand whilst also having alternative functions for example as transcriptional regulators in the nucleus. This raises new challenges for researchers. What are the cues/triggers that determine which role such proteins play? What are the trafficking pathways which regulate the spatial distribution of such proteins so that they can perform nuclear functions and under what circumstances are these alternative functions most relevant?

Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.