水稻RNA结合蛋白C3H12与RNA结合位点的鉴定

CCH型锌指蛋白质C3H12是进化上保守的RNA结合蛋白质,它含有5个串联的CCCH锌指结构域ZnF1-5,形成2个紧密的锌指簇ZnF1-3和ZnF4-5。早期的研究发现,C3H12可能通过与mRNA结合的方式在转录后水平调控基因的表达。然而,与C3H12结合的mRNA类型和他们的结合模式,并未通过实验得到证明。本文表达纯化了一系列C3H12截短及全长蛋白质,并合成了一些潜在RNA底物ARE9、ARE19及对照Random21。通过等温滴定量热法 (isothermal titration calorimetry, ITC) 确定了C3H12与富含腺嘌呤尿嘧啶单元 (AU-rich element, ARE) mRNA底物的结合,并揭示了互作核心区域和热力学性质。通过荧光光谱分析和微型热泳动 (microscale thermophoresis, MST)技术对ITC的结果进一步佐证。结果表明：(1) C3H12与ARE底物的相互作用是焓驱动的能量有利的 (ΔG<0) 特异性结合,结合比为1:1。(2) C3H12与ARE19的亲和力较ARE9更高（约2倍）。(3) C3H12中ZnF1-3在与ARE类底物的结合活性中发挥主导作用。(4) C3H12结构中的141个氨基酸残基的接头不直接参与和ARE底物的相互作用。本研究揭示的CCCH型锌指蛋白质C3H12与ARE底物结合模式,将为进一步在分子结构水平阐明C3H12与ARE底物结合的机制奠定基础。     

紫外交联合并串联亲和纯化高效鉴定蛋白复合物组分的RNA结合活性

RNA结合蛋白在RNA的生成与代谢中发挥着重要作用.我们在近年报道的PAR-CLIP(photoactivatableribonucleoside-enhanced crosslinking and immunoprecipitation)技术的基础上建立了一套快速、有效鉴定RNA结合蛋白的实验方法:以串联亲和纯化替代一步免疫沉淀获得高纯度蛋白-RNA复合物;将Sypro Ruby蛋白染色与RNA放射自显影相结合判断复合物中哪种或哪些组分为RNA结合蛋白,该方法命名为紫外交联合并的串联亲和纯化(cross-linkingand tandem affinity purification,CLiTAP).运用该方法对布氏锥虫的三种锌指蛋白ZC3H7、ZC3H34和ZC3H5进行分析,发现ZC3H7作为帽结合蛋白复合物的核心组分具有很强的RNA结合能力;ZC3H34结合RNA能力较弱,但其互作蛋白具有强的RNA结合活性;相比之下,ZC3H5及其复合物组分皆无RNA结合活性.这些结果表明,CLiTAP与蛋白质鉴定方法相结合,能够有效鉴定靶蛋白复合物中的RNA结合蛋白种类,也为进一步定位RNA结合位点、研究RNA结合蛋白的结构及作用机制奠定了基础.     

RNA结合蛋白与RNA互作鉴定技术

RNA结合蛋白（RNA binding proteins,RBPs）通过与RNA相互作用,广泛参与到RNA的剪切、转运、编辑、胞内定位及翻译调控等过程中。RNA领域尤其是非编码RNA（non-coding RNA,ncRNA）研究的快速发展,催生了多种RBPs RNAs相互作用鉴定技术。这些技术反之又推动了 RNA领域的研究进程。本文对紫外交联免疫沉淀（ultraviolet crosslinking and immunoprecipitation,CLIP）,CLIP cDNA文库高通量测序 (high-throughput sequencing of CLIP cDNA library,HITS-CLIP),光活化核苷增强的CLIP(photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation,PAR-CLIP),单核苷酸分离CLIP (individual nucleotide resolution CLIP,iCLIP),TRIBE (targets of RNA-binding protein identified by editing),RNA 标记,相互作用组捕获(interactome capture,IC) 和SerIC (serial RNA interactome capture)等RBPs-RNAs相互作用鉴定技术的基本原理和优缺点以及应用进行综述。     

快速展示糖苷水解酶活性架构单点突变导致结合力变化的电泳技术

酶分子在长期进化过程中形成一系列氨基酸残基组成的活性架构,参与底物的识别、结合与催化过程,而活性架构中相应氨基酸残基是如何影响酶分子结合底物的能力,进而影响酶分子的催化效率,一直是酶分子理性改造研究的热点.利用亲和电泳技术,可以快速展示内切纤维素酶Tr Cel12A和木聚糖酶Tl Xyn A活性架构中不同突变体的催化活性及其迁移率的变化,进而通过在不同底物浓度凝胶中蛋白质相对迁移率变化程度的定量回归分析,发现由氨基酸单点突变导致蛋白质迁移率的相对变化,可以定量表征酶分子突变前后结合底物能力的变化.亲和电泳测定的有效阻滞常数Kb值与等温滴定量热法和荧光光谱法测定的相关参数比较具有明显相关性.由于亲和电泳技术在测定酶分子与底物的结合能力时具有简便、快速、灵敏的特点,因而可作为常规生化实验室常规普筛技术来检测突变文库中系列突变体导致结合力的变化.     

RRM RNA结合蛋白的结构与功能

RRM RNA结合蛋白是一类含一个或数个RRM结构域及附属结构域的RNA结合蛋白,参与RNA前体的剪接、RNA的细胞定位、RNA的稳定性等多种转录后调控过程.在RRM基序中含有许多保守的氨基酸以保证对RNA的结合活性,但是这一家族的不同蛋白质却能特异地结合各种不同的RNA分子.RRM RNA结合蛋白与某些人类遗传性疾病及肿瘤相关.     

麦长管蚜唾液蛋白C002的基因克隆与RNA干扰研究

【目的】蚜虫唾液相关蛋白C002是一种水溶性唾液蛋白,在蚜虫取食过程中发挥着重要作用。本研究探讨了蚜虫唾液相关蛋白基因C002在麦长管蚜Sitobion avenae(F.)取食过程中的作用和功能。【方法】根据豌豆蚜基因C002序列,同源克隆了麦长管蚜C002基因,并应用荧光定量PCR和RNA干扰(RNAi)技术分别对该基因的表达规律和功能进行了研究。【结果】结果表明:麦长管蚜C002基因c DNA开放阅读框长663 bp,编码220个氨基酸,命名为Sv C002。转录水平的表达时序分析发现,Sv C002在蚜虫各个时期都有表达,在2龄若蚜时期表达量最高。ds RNA饲喂结果显示,麦长管蚜取食ds C002 4 d和6 d后,C002基因的表达量分别下降54%和69%,差异极显著(P<0.01),麦长管蚜存活率较对照组下降了32.2%和53.3%。将饲喂ds C002的麦长管蚜接种到感蚜小麦(北京837),蚜虫在短时期内出现大量死亡现象,第6天和第8天存活率分别为44.4%和35.6%,低于对照组并达到极显著水平。【结论】C002基因在麦长管蚜的取食行为中发挥着重要作用,可以作为麦长管蚜防治的潜在RNAi靶标基因。     

真核生物中锌指蛋白的结构与功能

真核生物中的许多蛋白质分子包含锌指结构区,这类蛋白称为锌指蛋白.锌指蛋白因其包含特殊的指状结构,在对DNA、蛋白质和RNA的识别和结合中起重要作用.许多锌指蛋白的锌指结构域包含能与DNA特异结合的区域,并与某些效应结构域（如KRAB、SCAN、BTB/POZ、SNAG、SANT和PLAG等）相连,这类锌指蛋白常作为转录因子起作用,可调控靶基因的转录.一些锌指蛋白包含蛋白质识别结构域（如LIM锌指、MYND锌指、PHD锌指和RING锌指等）,它们能够特异地介导蛋白质之间的相互作用,因此被称作蛋白适配器.此外,某些锌指蛋白还可以结合RNA,起转录后调控作用.本文就锌指蛋白与DNA、RNA以及蛋白质分子间的相互作用作一综述.     

RNA结合模体蛋白3对细胞总蛋白质合成的影响及其靶基因的鉴定

RNA结合模体蛋白3 (RNA binding motif protein 3, RBM3) 受低温应激产生,参与介导亚低温的神经保护功能,但其作用机制及其下游靶分子尚不清楚。本研究构建了人RBM3基因的重组表达质粒,运用一种新型的、非放射性方法即翻译表面感应 (surface sensing of translation, SUnSET) 来检测RBM3过表达对细胞总蛋白质合成(global protein synthesis, GPS)的影响。结果显示,RBM3过表达使细胞总蛋白质的合成水平上调23.7% (P < 0.001),这与RBM3过表达引发的真核翻译延伸因子2 (eukaryotic translation elongation factor 2, eEF2)及真核翻译起始因子2α (eukaryotic initiation factor 2α, eIF2α) 的活性增高相一致。对RBM3可能的下游靶基因内质网蛋白3 (reticulon 3,RTN3),以及Yes相关蛋白1 (Yes-associated protein 1,YAP1) 的表达进行分析。结果显示,RBM3使RTN3及YAP1在蛋白质水平的表达分别提高了51.7% (P < 0.01) 与43.3% (P < 0.01)。与蛋白质水平变化相比,RBM3使YAP1在mRNA水平上调了2.0倍 (P < 0.001),但对RTN3的mRNA表达未见显著影响。以上研究表明,SUnSET是一种稳定、可靠的细胞GPS的检测手段;RBM3可显著促进细胞GPS,且对其下游基因RTN3和YAP1存在靶向关系。本研究的结果为深入探讨RBM3的神经保护作用机制提供了理论基础。     

一种改良的热酚法高效快速提取酿酒酵母总RNA

以酿酒酵母单倍体CEN.PK1与双倍体CEN.PK2为研究对象,利用改良热酚法,快速高效地提取酿酒酵母总RNA.结果显示应用该方法提取的酿酒酵母CEN.PK1和CEN.PK2的总RNA经分光光度计测定浓度为7.63 μg/μL和3.77 μg/μL,OD260/280分别为2.10和2.04,OD260/230分别为2.32和2.24,浓度与纯度均能达到后续分子生物学试验的要求.经PCR与荧光定量PCR检测,该方法提取的RNA无DNA污染,可作为PCR反应的模板,与Trizol方法提取RNA相比,该方法不仅浓度高并且可将提取时间由常规的2.5 h缩短为1.5 h,具有操作简单、高质、高效等优点,经多次试验证明,此方法同样适用于酿酒酵母总RNA的大量提取试验,有较高的实用价值.     

水稻双链RNA结合蛋白同源基因OsRBP的克隆及其表达的分析

在基因数据库中发现两个水稻EST片段与大白菜BcpLH基因的双链RNA结合结构域 (dsRBD)有同源的区域 ,根据同源片段的位置特征设计引物 ,用RT-RCR扩增粳稻 (Oryzasativasubsp .japonica)愈伤组织的cDNA ,得到大小为 1.8kb的DNA片段。该cDNA片段含完整的编码区 ,有两个典型的dsRBD ,分别与BcpLH的dsRBD在氨基酸水平上同源性为 75 %左右 ,故将其命名为OsRBP。RT -PCR表达分析显示该基因在未成熟的种子和愈伤组织中表达 ,在根、茎、叶、穗、成熟种子及胚芽鞘中没有表达信号 ,由此推测该基因的表达可能与种子和胚的早期发育相关。该研究首次从水稻中分离到双链RNA结合蛋白基因 ,并初步研究了其表达方式 ,为进一步探讨水稻重要器官的发育和植物中双链RNA结合蛋白的调节作用奠定了基础     

LRP16结合poly(ADP-ribose)关键位点的识别

LRP16作为macro domain 家族成员,可识别、结合poly(ADP-ribose),参与DNA损伤修复的早期反应. 但究竟LRP16通过其何种氨基酸位点识别、结合poly(ADP ribose)（PAR）尚不十分清楚．本研究首先通过对LRP16蛋白的结构分析,查找LRP16结合PAR的候选氨基酸位点,然后利用丙氨酸扫描技术构建系列LRP16（点）突变体, 并且进行原核蛋白表达与纯化,将获得的蛋白质进行斑点杂交实验,检测LRP16突变体蛋白与PAR的结合活性.测序结果显示,LRP16点突变基因序列成功插入到原核表达载体pGEX-6p-1中|斑点杂交实验显示,LRP16中第160位D和第161位I突变成A后,其与PAR结合能力明显减弱,而第181位G、183位V、184位D同时突变为A,LRP16与PAR的结合活性有部分减弱. 结果表明,LRP16中第160位和第161位的氨基酸是其与PAR结合的关键位点.     

Identification of a Unique Fe-S Cluster Binding Site in a Glycyl-Radical Type Microcompartment Shell Protein

Recently, progress has been made toward understanding the functional diversity of bacterial microcompartment (MCP) systems, which serve as protein-based metabolic organelles in diverse microbes. New types of MCPs have been identified, including the glycyl-radical propanediol (Grp) MCP. Within these elaborate protein complexes, BMC-domain shell proteins [bacterial microcompartment (in reference to the shell protein domain)] assemble to form a polyhedral barrier that encapsulates the enzymatic contents of the MCP. Interestingly, the Grp MCP contains a number of shell proteins with unusual sequence features. GrpU is one such shell protein whose amino acid sequence is particularly divergent from other members of the BMC-domain superfamily of proteins that effectively defines all MCPs. Expression, purification, and subsequent characterization of the protein showed, unexpectedly, that it binds an iron-sulfur cluster. We determined X-ray crystal structures of two GrpU orthologs, providing the first structural insight into the homohexameric BMC-domain shell proteins of the Grp system. The X-ray structures of GrpU, both obtained in the apo form, combined with spectroscopic analyses and computational modeling, show that the metal cluster resides in the central pore of the BMC shell protein at a position of broken 6-fold symmetry. The result is a structurally polymorphic iron-sulfur cluster binding site that appears to be unique among metalloproteins studied to date.     

Identification of Target Binding Site in Photoreceptor Guanylyl Cyclase-activating Protein 1 (GCAP1)

Retinal guanylyl cyclase (RetGC)-activating proteins (GCAPs) regulate visual photoresponse and trigger congenital retinal diseases in humans, but GCAP interaction with its target enzyme remains obscure. We mapped GCAP1 residues comprising the RetGC1 binding site by mutagenizing the entire surface of GCAP1 and testing the ability of each mutant to bind RetGC1 in a cell-based assay and to activate it in vitro. Mutations that most strongly affected the activation of RetGC1 localized to a distinct patch formed by the surface of non-metal-binding EF-hand 1, the loop and the exiting helix of EF-hand 2, and the entering helix of EF-hand 3. Mutations in the binding patch completely blocked activation of the cyclase without affecting Ca²⁺ binding stoichiometry of GCAP1 or its tertiary fold. Exposed residues in the C-terminal portion of GCAP1, including EF-hand 4 and the helix connecting it with the N-terminal lobe of GCAP1, are not critical for activation of the cyclase. GCAP1 mutants that failed to activate RetGC1 in vitro were GFP-tagged and co-expressed in HEK293 cells with mOrange-tagged RetGC1 to test their direct binding in cyto. Most of the GCAP1 mutations introduced into the “binding patch” prevented co-localization with RetGC1, except for Met-26, Lys-85, and Trp-94. With these residues mutated, GCAP1 completely failed to stimulate cyclase activity but still bound RetGC1 and competed with the wild type GCAP1. Thus, RetGC1 activation by GCAP1 involves establishing a tight complex through the binding patch with an additional activation step involving Met-26, Lys-85, and Trp-94.     

Changes in [3H]Forskolin Binding to Adenylate Cyclase and [3H]Phorbol Dibutyrate Binding to Protein Kinase C in Pentobarbital Tolerant/Dependent Rats

These studies were designed to examine the effect of chronic administration of pentobarbital on activity of adenylate cyclase (AC) and protein kinase C (PKC) in the rat brain by autoradiography. Recently, it has been suggested that the phosphorylation of specific proteins may be involved in the development of physical dependence. An experimental model of barbiturate tolerance and dependence was developed using i.c.v. infusion of pentobarbital (300 g/ 10 l/hr for 7 days) by osmotic minipumps and abrupt withdrawal from pentobarbital. The levels of [³H]forskolin binding were elevated (28–67%) in cortex, thalamus, dentate gyrus, hippocampal CA3 and cerebellum of the pentobarbital withdrawal animals, while these changes were not observed in tolerant rats. The levels of [³H]phorbol dibutyrate binding were highly elevated (38–65%) in the region of cortex, caudate putamen, septum, thalamus, dentate gyrus, and cerebellum of rats withdrawal from pentobarbital. These results show that the levels of AC and PKC were significantly elevated in pentobarbital withdrawal rats, and suggest that the levels of AC and PKC are altered in a region-specific manner during pentobarbital withdrawal.     

Identification of Bilirubin Binding Site in Type I Collagen

The interaction of bilirubin with collagen in the significance of jaundice incidence have been previously reported and investigated. The novel peptide sequences containing bilirubin binding domain was identified and located to develop a basis for further studies investigating the interactions of collagen with bilirubin in the present study. In this study an intricate interaction between bilirubin and collagen was characterized and their binding domain has been established using in-gel digestion and LC–MS/MS analysis based on the collagen sequencing and peptide mass fingerprinting. The biotinylated bilirubin derivatives bind to α₁(I) chain but not to α₂(I) chains which clearly designates that bilirubin shows greater affinity to α₁ chains of collagen. The intact proteins collected after analyzing the resulting complex mixture of peptides was used for peptide mapping. Using the electrospray method, among the other peptide sequence information obtained, the molecular weight of collagen alpha-2(I) chain was obtained by locating a 130 kDa weight peptide sequences with greater pi value (9.14) with 1,364 amino acid residues and collagen alpha-1(I) chain with 1,463 amino acid residues with 138.9 kDa molecular weight. This information leads to locate the exact sequence of these helices focussing on the domain identification. The total charge of the peptide domain sequences infers that the bilirubin participates in the electrostatic mode of interaction with collagen peptide. Moreover, other modes of interactions such as hydrogen bonding, covalent interactions and hydrophobic interactions are possible.     

Mutant Ribosomal Protein with Defective RNA Binding Site

THE 30S ribosomal subunits of Escherichia coli contain twenty-one different proteins^1–4, which together with 16S RNA can reassemble in vitro to form functional 30S particles⁵. Five proteins can individually bind to specific sites on the 16S RNA^6–8 and these are S4, S7, S8, S15 and S20 (in the nomenclature recently adopted by several laboratories to report results with the E. coli system⁹). We report here the first identification of a mutation that affects a ribosomal protein-nucleic acid interaction.     

Upregulation of TPX2 by STAT3: Identification of a Novel STAT3 Binding Site

TPX2, a protein involved in mitosis, is considered a good marker for actively proliferating tissues, highly expressed in a number of cancer cells. We show the presence of high-affinity binding site for STAT3 in the 5′-flanking region of the Tpx2 gene, which is in vivo bound by activated STAT3. A specific STAT3 peptide inhibitor represses the expression of the Tpx2 gene and inhibits the binding of STAT3 to its consensus sequence in human cell lines where STAT3 is activated. These results indicate that activated STAT3 contributes to the over-expression of Tpx2 through the binding to an enhancer site.     

Identification of VPS13C as a Galectin-12-Binding Protein That Regulates Galectin-12 Protein Stability and Adipogenesis

Galectin-12, a member of the galectin family of β-galactoside-binding animal lectins, is preferentially expressed in adipocytes and required for adipocyte differentiation in vitro. This protein was recently found to regulate lipolysis, whole body adiposity, and glucose homeostasis in vivo. Here we identify VPS13C, a member of the VPS13 family of vacuolar protein sorting-associated proteins highly conserved throughout eukaryotic evolution, as a major galectin-12-binding protein. VPS13C is upregulated during adipocyte differentiation, and is required for galectin-12 protein stability. Knockdown of Vps13c markedly reduces the steady-state levels of galectin-12 by promoting its degradation through primarily the lysosomal pathway, and impairs adipocyte differentiation. Our studies also suggest that VPS13C may have a broader role in protein quality control. The regulation of galectin-12 stability by VPS13C could potentially be exploited for therapeutic intervention of obesity and related metabolic diseases.     

Drug Promiscuity in PDB: Protein Binding Site Similarity Is Key

Drug repositioning applies established drugs to new disease indications with increasing success. A pre-requisite for drug repurposing is drug promiscuity (polypharmacology) – a drug’s ability to bind to several targets. There is a long standing debate on the reasons for drug promiscuity. Based on large compound screens, hydrophobicity and molecular weight have been suggested as key reasons. However, the results are sometimes contradictory and leave space for further analysis. Protein structures offer a structural dimension to explain promiscuity: Can a drug bind multiple targets because the drug is flexible or because the targets are structurally similar or even share similar binding sites? We present a systematic study of drug promiscuity based on structural data of PDB target proteins with a set of 164 promiscuous drugs. We show that there is no correlation between the degree of promiscuity and ligand properties such as hydrophobicity or molecular weight but a weak correlation to conformational flexibility. However, we do find a correlation between promiscuity and structural similarity as well as binding site similarity of protein targets. In particular, 71% of the drugs have at least two targets with similar binding sites. In order to overcome issues in detection of remotely similar binding sites, we employed a score for binding site similarity: LigandRMSD measures the similarity of the aligned ligands and uncovers remote local similarities in proteins. It can be applied to arbitrary structural binding site alignments. Three representative examples, namely the anti-cancer drug methotrexate, the natural product quercetin and the anti-diabetic drug acarbose are discussed in detail. Our findings suggest that global structural and binding site similarity play a more important role to explain the observed drug promiscuity in the PDB than physicochemical drug properties like hydrophobicity or molecular weight. Additionally, we find ligand flexibility to have a minor influence.